Cationic lipid

ABSTRACT

The present invention provides a cationic lipid, which allow nucleic acids to be easily introduced into cells, represented by formula (I) 
     (wherein: R 1  and R 2  are, the same or different, alkenyl, etc, and
 
X 1  and X 2  are hydrogen atoms, or are combined together to form a single bond or alkylene, and
 
X 3  is absent or is alkyl, etc,
 
Y is absent or anion,
 
a and b are, the same or different, 0 to 3, and
 
L 3  is a single bond, etc,
 
R 3  is alkyl, etc,
 
L 1  and L 2  are —O—, —CO—O— or —O—CO—),
 
a composition comprising the cationic lipid and a nucleic acid, and
 
a method for introducing a nucleic acid into a cell by using the composition comprising the cationic lipid and the nucleic acid, and the like.

TECHNICAL FIELD

The present invention relates to a novel cationic lipid that allows, forexample, nucleic acid to be easily introduced into cells, and to a novelcomposition comprising the cationic lipid, and the like.

BACKGROUND ART

Cationic lipids are amphiphilic molecules that generally contain alipophilic region containing one or more hydrocarbon groups, and ahydrophilic region containing at least one positively charged polar headgroup. Cationic lipids are useful, because cationic lipids facilitateentry of macromolecules such as nucleic acids into the cytoplasm throughthe cell plasma membrane by forming a positively charged (total charge)complex with macromolecules such as nucleic acids. This process,performed in vitro and in vivo, is known as transfection.

Typically, cationic lipids are used either alone, or in combination withneutral lipids such as phospholipids. A combination of cationic lipidsand neutral lipids is known to be useful, because it can easily form avesicle that contains an aligned lipid bilayer. Vesicles and liposomesformed by cationic lipids either alone or in combination with neutrallipids have many positive charges on the surface, and, with thesecharges, can form a complex with polynucleotides or other anionicmolecules such as negatively charged proteins. The remaining totalcationic charge on the surface of a polynucleotide/cationiclipid/neutral lipid complex can cause strong interaction with the cellmembrane, mainly with the negative charge on the surface of the cellmembrane.

To date, many different cationic lipids have been synthesized fortransfection, and are commercially available. Such cationic lipidsinclude, for example, Lipofectin, Lipofectin ACE, Lipofect AMINE,Transfeactam, DOTAP, etc.

The N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride(DOTMA), etc disclosed in Patent Document 1 are one of the cationiclipids developed in the early. DOTMA etc. are characterized by thepropanaminium group having quaternary nitrogen providing a cationic partto the molecule, and a pair of higher hydrocarbons attached to thepropyl backbone of the molecule by an ether bond. The quaternarynitrogen is trisubstituted with relatively short alkyl chains such asmethyl groups. As structurally similar cationic lipid,N-(2,3-di-(9-(Z)-octadecenoyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTAP) contains acyl groups, instead of the ether-bonded alkylgroups.

For example, theN-[1-(2,3-dioleyloxypropyl)]-N,N-dimethyl-N-hydroxyethylammonium bromide(DORIE), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate(DOSPA), etc disclosed in Patent Documents 2 and 3 are characterized bythe propanaminium group having quaternary nitrogen providing a cationicpart to the molecule, and a pair of higher hydrocarbons attached to thepropyl backbone of the molecule by an ether bond, the propanaminiumgroup. The quaternary nitrogen is characterized by being trisubstitutedwith relatively short alkyl chains such as methyl groups, and withhydroxyalkyl.

Patent Document 4 discloses, for example,1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), etc. DLinDMA, etcare characterized by the higher alkyl group that contains at least twounsaturated moieties. The higher alkyl group is contained as areplacement for the higher alkyl groups of the structurally similarcationic lipids DOTAP and DOTMA for the purpose of developing moreflexible cationic lipids and improving the membrane fluidity ofliposomes or the like. Patent Document 5 discloses, for example,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolan (DLin-K-DMA), etc.

CITATION LIST Patent Documents

[Patent Document 1] Japanese Published Unexamined Patent Application No.161246/1986 (U.S. Pat. No. 5,049,386)

[Patent Document 2] WO1991/16024

[Patent Document 3] WO1997/019675

[Patent Document 4] WO2005/121348

[Patent Document 5] WO2009/086558

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a novel cationic lipidthat allows, for example, nucleic acids to be easily introduced intocells, and a novel composition comprising the cationic lipid, and thelike.

Means for Solving the Problems

The present invention is concerned with the following (1) to (27).

(1) A cationic lipid represented by formula (I):

(wherein:R¹ and R² are, the same or different, each linear or branched alkyl,alkenyl or alkynyl having 12 to 24 carbon atoms, or R¹ and R² arecombined together to form dialkylmethylene, dialkenylmethylene,dialkynylmethylene or alkylalkenylmethylene, X¹ and X² are hydrogenatoms, or are combined together to form a single bond or alkylene, X³ isabsent or is alkyl having 1 to 6 carbon atoms, or alkenyl having 3 to 6carbon atoms,

when X³ is absent,

-   -   Y is absent, a and b are 0, L³ is a single bond, R³ is alkyl        having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms,        pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having        1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms        substituted with 1 to 3 substituent(s), which is(are), the same        or different, amino, monoalkylamino, dialkylamino,        trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl,        dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, and L¹        and L² are —O—,    -   Y is absent, a and b are, the same or different, 0 to 3, and are        not 0 at the same time, L³ is a single bond, R³ is alkyl having        1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms,        pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having        1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms        substituted with 1 to 3 substituent(s), which is(are), the same        or different, amino, monoalkylamino, dialkylamino,        trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl,        dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, L¹ and        L² are, the same or different, —O—, —CO—O— or —O—CO—,    -   Y is absent, a and b are, the same or different, 0 to 3, L³ is a        single bond, R³ is a hydrogen atom, and L¹ and L² are, the same        or different, —O—, —CO—O— or —O—CO—, or    -   Y is absent, a and b are, the same or different, 0 to 3, L³ is        —CO— or —CO—O—, R³ is pyrrolidin-2-yl, pyrrolidin-3-yl,        piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl,        morpholin-3-yl, or alkyl having 1 to 6 carbon atoms or alkenyl        having 3 to 6 carbon atoms substituted with 1 to 3        substituent(s), which is(are), the same or different, amino,        monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,        carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl,        piperidyl or morpholinyl, wherein at least one of the        substituents is amino, monoalkylamino, dialkylamino,        trialkylammonio, pyrrolidinyl, piperidyl or morpholinyl, and L¹        and L² are, the same or different, —O—, —CO—O— or —O—CO—, and

when X³ is alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6carbon atoms,

-   -   Y is a pharmaceutically acceptable anion, a and b are, the same        or different, 0 to 3, L³ is a single bond, R³ is alkyl having 1        to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms,        pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having        1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms        substituted with 1 to 3 substituent(s), which is(are), the same        or different, amino, monoalkylamino, dialkylamino,        trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl,        dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, L¹ and        L² are, the same or different, —O—, —CO—O— or —O—CO—).

(2) The cationic lipid as set forth above in (1), wherein L¹ and L² are—O— or —O—CO—, and R¹ and R² are dodecyl, tetradecyl, hexadecyl,octadecyl, icosyl, docosyl, tetracosyl, (Z)-tetradec-9-enyl,(Z)-hexadec-9-enyl, (Z)-octadec-6-enyl, (Z)-octadec-9-enyl,(E)-octadec-9-enyl, (Z)-octadec-1-enyl, (9Z,12Z)-octadec-9,12-dienyl,(9Z,12Z,15Z)-octadec-9,12,15-trienyl, (Z)-icos-11-enyl,(11Z,14Z)-icos-11,14-dienyl, 3,7,11-trimethyldodeca-2,6,10-trienyl or3,7,11,15-tetramethylhexadec-2-enyl.

(3) The cationic lipid as set forth above in (1), wherein L¹ and L² are—CO—O—, and R¹ and R² are tridecyl, pentadecyl, heptadecyl, nonadecyl,heneicosyl, tricosyl, (Z)-tridec-8-enyl, (Z)-pentadec-8-enyl,(Z)-heptadec-5-enyl, (Z)-heptadec-8-enyl, (E)-heptadec-8-enyl,(Z)-heptadec-10-enyl, (8Z,11Z)-heptadec-8,11-dienyl,(8Z,11Z,14Z)-octadec-8,11,14-trienyl, (Z)-nonadec-10-enyl,(10Z,13Z)-nonadec-10,13-dienyl, (11Z,14Z)-icos-11,14-dienyl,2,6,10-trimethylundec-1,5,9-trienyl or2,6,10,14-tetramethylpentadec-1-enyl.

(4) The cationic lipid as set forth above in any one of (1) to (3),wherein a and b are both 0 or 1.

(5) The cationic lipid as set forth above in any one of (1) to (4),wherein L³ is a single bond, R³ is a hydrogen atom, methyl,pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having 1 to 6carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to3 substituent(s), which is(are), the same or different, amino,monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidylor morpholinyl, and L and L² are —O—.

(6) The cationic lipid as set forth above in any one of (1) to (5),wherein L³ is —CO— or —CO—O—, R³ is pyrrolidin-3-yl, piperidin-3-yl,piperidin-4-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are),the same or different, amino, monoalkylamino, dialkylamino,trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl,dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, wherein atleast one of the substituents is amino, monoalkylamino, dialkylamino,trialkylammonio, pyrrolidinyl, piperidyl or morpholinyl, and L¹ and L²are identically —CO—O— or —O—CO—.

(7) The cationic lipid as set forth above in any one of (1) to (6),wherein X¹ and X² are combined together to form a single bond oralkylene.

(8) The cationic lipid as set forth above in any one of (1) to (5),wherein X¹ and X² are combined together to form a single bond oralkylene, and R³ is a hydrogen atom, methyl, or alkyl having 1 to 6carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to3 substituent(s), which is(are), the same or different, amino, hydroxyor carbamoyl.

(9) The cationic lipid as set forth above in any one of (1) to (5),wherein X¹ and X² are hydrogen atoms, and R³ is a hydrogen atom, methyl,or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbonatoms substituted with 1 to 3 substituent(s), which is(are), the same ordifferent, amino, hydroxy or carbamoyl.

(10) The cationic lipid as set forth above in (6), wherein X¹ and X² arecombined together to form a single bond or alkylene, and R³ is alkylhaving 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atomssubstituted with 1 to 3 substituent(s), which is(are), the same ordifferent, amino, hydroxy or carbamoyl.

(11) The cationic lipid as set forth above in (6), wherein X¹ and X² arehydrogen atoms, and R³ is alkyl having 1 to 6 carbon atoms or alkenylhaving 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), whichis(are), the same or different, amino, hydroxy or carbamoyl.

(12) The cationic lipid as set forth above in any one of (1) to (11),wherein X³ is absent or is methyl.

(13) A composition that comprises the cationic lipid as set forth abovein any one of (1) to (12), and a nucleic acid.

(14) A composition comprising a complex particle of the cationic lipidas set forth above in any one of (1) to (12) and a nucleic acid, or acomplex particle of a nucleic acid and a combination of the cationiclipid and a neutral lipid and/or a polymer.

(15) A composition comprising a lipid particle constituted of a complexparticle of the cationic lipid as set forth above in any one of (1) to(12) and a nucleic acid, or a complex particle of a nucleic acid and acombination of the cationic lipid and a neutral lipid and/or a polymer,and a lipid membrane that encapsulates the complex particle.

(16) The composition as set forth above in any one of (13) to (15),wherein the nucleic acid is a nucleic acid having an activity ofsuppressing the expression of the target gene by utilizing RNAinterference (RNAi).

(17) The composition as set forth above in (16), wherein the target geneis a gene associated with tumor or inflammation.

(18) A method for introducing the nucleic acid into a cell by using thecomposition of any one as set forth above in (14) to (17).

(19) The method as set forth above in (18), wherein the cell is a cellat a tumor or inflammation site of a mammal.

(20) The method as set forth above in (18) or (19), wherein the cell isa cell in the liver, lungs, kidneys or spleen of a mammal.

(21) The method as set forth above in (19) or (20), wherein the methodof the introduction into a cell is a method of introduction into a cellby intravenous administration.

(22) A method for treating cancer or inflammatory disease, the methodincluding administering the composition as set forth above in (17) to amammal.

(23) The method as set forth above in (22), wherein the method ofadministration is intravenous administration.

(24) A medicament comprising the composition as set forth above in (16)and for treating disease by administration of the composition to amammal.

(25) The medicament as set forth above in (24), wherein the method ofadministration is intravenous administration.

(26) A cancer or inflammatory disease therapeutic agent comprising thecomposition as set forth above in (17) and for treating cancer orinflammatory disease by administration of the composition to a mammal.

(27) The cancer or inflammatory disease therapeutic agent as set forthabove in (26), wherein the method of administration is intravenousadministration.

Advantage of the Invention

A composition comprising the novel cationic lipid of the presentinvention and a nucleic acid can be administered to mammals, etc and,for example, the like to easily introduce the nucleic acid into cellsand the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 1 to 10) into human liver cancer-derived cell lineHepG2. The vertical axis represents target gene mRNA expression raterelative to the negative control taken at 1; the horizontal axisrepresents nucleic acid concentration (nM), and the compound numbers ofthe cationic lipids used.

FIG. 2 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 11-20) into cells as that in FIG. 1.

FIG. 3 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 21 to 30) into cells as that in FIG. 1.

FIG. 4 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 31 to 37) into cells as that in FIG. 1.

FIG. 5 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 38 to 48) into cells as that in FIG. 1.

FIG. 6 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 49 to 58) into cells as that in FIG. 1.

FIG. 7 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 59 to 68) into cells as that in FIG. 1.

FIG. 8 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 69 to 79) into cells as that in FIG. 1.

FIG. 9 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 80 to 90) into cells as that in FIG. 1.

FIG. 10 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 91 to 100) into cells as that in FIG. 1.

FIG. 11 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 101 to 110) into cells as that in FIG. 1.

FIG. 12 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Example 118 (preparationsusing compounds 111 to 115) into cells as that in FIG. 1.

FIG. 13 shows the expression rate of target gene mRNA after theintroduction of the preparations obtained in Comparative Examples 1 to 8into cells as that in FIG. 1.

MODE FOR CARRYING OUT THE INVENTION

A cationic lipid of the present invention is represented by thefollowing formula (I):

(wherein:R¹ and R² are, the same or different, each linear or branched alkyl,alkenyl or alkynyl having 12 to 24 carbon atoms, or R¹ and R² arecombined together to form dialkylmethylene, dialkenylmethylene,dialkynylmethylene or alkylalkenylmethylene, X¹ and X² are hydrogenatoms, or are combined together to form a single bond or alkylene, X³ isabsent or represents alkyl having 1 to 6 carbon atoms, or alkenyl having3 to 6 carbon atoms,

when X³ is absent,

-   -   Y is absent, a and b are 0, L³ is a single bond, R³ is alkyl        having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms,        pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having        1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms        substituted with 1 to 3 substituent(s), which is(are), the same        or different, amino, monoalkylamino, dialkylamino,        trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl,        dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, and L¹        and L² are —O—,    -   Y is absent, a and b are, the same or different, 0 to 3, and are        not 0 at the same time, L³ is a single bond, R³ is alkyl having        1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms,        pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having        1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms        substituted with 1 to 3 substituent(s), which is(are), the same        or different, amino, monoalkylamino, dialkylamino,        trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl,        dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, L¹ and        L² are, the same or different, —O—, —CO—O— or —O—CO—,    -   Y is absent, a and b are, the same or different, 0 to 3, L³ is a        single bond, R³ is a hydrogen atom, and L¹ and L² are, the same        or different, —O—, —CO—O— or —O—CO—, or    -   Y is absent, a and b are, the same or different, 0 to 3, L³ is        —CO— or —CO—O—, R³ is pyrrolidin-2-yl, pyrrolidin-3-yl,        piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl,        morpholin-3-yl, or alkyl having 1 to 6 carbon atoms or alkenyl        having 3 to 6 carbon atoms substituted with 1 to 3        substituent(s), which is(are), the same or different, amino,        monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,        carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl,        piperidyl or morpholinyl, wherein at least one of the        substituents is amino, monoalkylamino, dialkylamino,        trialkylammonio, pyrrolidinyl, piperidyl or morpholinyl, and L¹        and L² are, the same or different, —O—, —CO—O— or —O—CO—, and

when X³ is alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6carbon atoms,

-   -   Y is a pharmaceutically acceptable anion, a and b are, the same        or different, 0 to 3, L³ is a single bond, R³ is alkyl having 1        to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms,        pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl,        piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl,        or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6        carbon atoms substituted with 1 to 3 substituent(s), which        is(are), the same or different, amino, monoalkylamino,        dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl,        monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or        morpholinyl, L¹ and L² are, the same or different, —O—, —CO—O—        or —O—CO—).

The compound represented by the formula (I) will be hereinafter alsoreferred to as “Compound (I)”. The same is also applicable to compoundsdesignated with other numbers.

In the definition of each group in formula (I), examples of the linearor branched alkyl having 12 to 24 carbon atoms include dodecyl,tridecyl, tetradecyl, 2,6,10-trimethylundecyl, pentadecyl,3,7,11-trimethyldodecyl, hexadecyl, heptadecyl, octadecyl,6,10,14-trimethylpentadecan-2-yl, nonadecyl,2,6,10,14-tetramethylpentadecyl, icosyl, 3,7,11,15-tetramethylhexadecyl,heneicosyl, docosyl, tricosyl, tetracosyl, and the like.

The linear or branched alkenyl having 12 to 24 carbon atoms may be alinear or branched alkenyl having 12 to 24 carbon atoms and having 1 to3 double bonds. Examples thereof include (Z)-tridec-8-enyl,(Z)-tetradec-9-enyl, (Z)-pentadec-8-enyl, (Z)-hexadec-9-enyl,(Z)-heptadec-5-enyl, (Z)-octadec-6-enyl, (Z)-heptadec-8-enyl,(Z)-octadec-9-enyl, (E)-heptadec-8-enyl, (E)-octadec-9-enyl,(Z)-heptadec-10-enyl, (Z)-octadec-11-enyl,(8Z,11Z)-heptadeca-8,11-dienyl, (9Z,12Z)-octadeca-9,12-dienyl,(8Z,11Z,14Z)-octadeca-8,11,14-trienyl,(9Z,12Z,15Z)-octadeca-9,12,15-trienyl, (Z)-nonadec-10-enyl,(Z)-icos-11-enyl, (10Z,13Z)-nonadeca-10,13-dienyl,(1Z,14Z)-icosa-11,14-dienyl, 2,6,10-trimethylundeca-1,5,9-trienyl,3,7,11-trimethyldodeca-2,6,10-trienyl,2,6,10,14-tetramethylpentadec-1-enyl, and3,7,11,15-tetramethylhexadec-2-enyl. Of these, (Z)-pentadec-8-enyl,(Z)-hexadec-9-enyl, (Z)-heptadec-5-enyl, (Z)-octadec-6-enyl,(Z)-heptadec-8-enyl, (Z)-octadec-9-enyl, (8Z,11Z)-heptadeca-8,11-dienyl,(9Z,12Z)-octadeca-9,12-dienyl, and the like are preferable.

The linear or branched alkynyl having 12 to 24 carbon atoms may be alinear or branched alkynyl having 12 to 24 carbon atoms and having 1 to3 triple bonds. Examples thereof include dodec-11-ynyl, tridec-12-ynyl,pentadec-6-ynyl, hexadec-7-ynyl, pentadeca-4,6-diynyl,hexadeca-5,7-diynyl, heptadec-8-ynyl, and octadec-9-ynyl.

Examples of the alkylene include methylene, ethylene, propylene, and thelike.

Examples of the alkyl having 1 to 6 carbon atoms include methyl, ethyl,propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl,cyclobutyl, cyclopropylmethyl, pentyl, isopentyl, sec-pentyl, neopentyl,tert-pentyl, cyclopentyl, hexyl, and cyclohexyl. Of these, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, hexyl, and thelike are preferable, with methyl, ethyl, propyl, and the like being morepreferable.

Examples of the alkenyl having 3 to 6 carbon atoms include allyl,1-propenyl, butenyl, pentenyl, and hexenyl. Of these, allyl or the likeis preferable.

The alkyl moiety in the substituted alkyl having 1 to 6 carbon atoms andthe alkenyl moiety in the substituted alkenyl having 3 to 6 carbon atomshave the same definitions of the alkyl having 1 to 6 carbon atoms andthe alkenyl having 3 to 6 carbon atoms as described above, respectively.

The alkyl, alkenyl, and alkynyl moieties in the dialkylmethylene,dialkenylmethylene, dialkynylmethylene or alkylalkenylmethylene have thesame definitions as the linear or branched alkyl having 12 to 24 carbonatoms, the linear or branched alkenyl having 12 to 24 carbon atoms, andthe linear or branched alkynyl having 12 to 24 carbon atoms,respectively. In addition, it is further preferable that thedialkylmethylene, dialkenylmethylene, and dialkynylmethylene have thesame alkyl, alkenyl, and alkynyl moieties, respectively.

In the present invention, examples of the pharmaceutically acceptableanions include inorganic ions such as chloride ions, bromide ions,nitric acid ions, sulfuric acid ions, and phosphoric acid ions, organicacid ions such as acetic acid ions, oxalic acid ions, maleic acid ions,fumaric acid ions, citric acid ions, benzoic acid ions, andmethanesulfonic acid ions, and the like.

In the present invention, each of pyrrolidin-2-yl, pyrrolidin-3-yl,piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, andmorpholin-3-yl includes one in which the hydrogen atom bonded on thenitrogen atom in the ring is converted into methyl or ethyl.

Each of the monoalkylamino and the dialkylamino may be an amino which issubstituted with one or two alkyls, being the same or different, andhaving a carbon number of 1 to 6 (having the same definition as above)or an alkyl or alkyls having a carbon number of 1 to 6 (having the samedefinition as above) substituted with amino, methylamino, ethylamino,dimethylamino, diethylamino, pyrrolidinyl, piperidyl or morpholinyl.Examples thereof include methylamino, ethylamino, propylamino,butylamino, pentylamino, hexylamino, dimethylamino, diethylamino,ethylmethylamino, methylpropylamino, butylmethylamino,methylpentylamino, hexylmethylamino, aminoethylamino, aminopropylamino,(aminoethyl)methylamino, and bis(aminoethyl)amino. Of these,methylamino, ethylamino, dimethylamino, diethylamino, aminopropylamino,and bis(aminoethyl)amino, and the like are preferable.

In the present invention, the amino, monoalkylamino, and dialkylaminomay form ammonio, monoalkylammonio, and dialkylammonio, respectively,through coordination of a hydrogen ion to a lone pair on the nitrogenatom. The amino, monoalkylamino, and dialkylamino include ammonio,monoalkylammonio, and dialkylammonio, respectively.

The trialkylammonio may be an ammonio substituted with threesubstituents, which are, the same or different, alkyl having 1 to 6carbon atoms (having the same definition as described above), and alkylhaving 1 to 6 carbon atoms (having the same definition as describedabove) substituted with amino, methylamino, ethylamino, dimethylamino,diethylamino, pyrrolidinyl, piperidyl or morpholinyl. Examples thereofinclude trimethylammonio, ethyldimethylammonio, diethylmethylammonio,triethylammonio, tripropylammonio, tributylammonio, tripentylammonio,trihexylammonio, tris(aminoethyl)ammonio, (aminoethyl)dimethylammonio,bis(aminoethyl)methylammonio, and the like. Preferred examples thereofinclude trimethylammonio, triethylammonio, tris(aminoethyl)ammonio,(aminoethyl)dimethylammonio, bis(aminoethyl)methylammonio, and the like.

In the present invention, the ammonio, monoalkylammonio, anddialkylammonio in which a hydrogen ion coordinates to a lone pair on thenitrogen atom of the amino, monoalkylamino, and dialkylamino,respectively, and the trialkylammonio may form salts withpharmaceutically acceptable anions (having the same definitions asdescribed above).

The alkoxy may be hydroxy which is substituted with an alkyl having acarbon number of 1 to 6 (having the same definition as above) or analkyl having a carbon number of 1 to 6 (having the same definition asabove) substituted with amino, methylamino, ethylamino, dimethylamino,diethylamino, pyrrolidinyl, piperidyl or morpholinyl. Examples thereofinclude methoxy, ethoxy, propyloxy, butyloxy, pentyloxy, hexyloxy,aminoethoxy, and methylaminoethoxy. Of these, methoxy, ethoxy,aminoethoxy, methylaminoethoxy, and the like are preferable.

The monoalkylcarbamoyl and dialkylcarbamoyl may be carbamoylssubstituted with one or two substituent(s), which is(are), the same ordifferent, alkyl having 1 to 6 carbon atoms (having the same definitionas described above), and alkyl having 1 to 6 carbon atoms (having thesame definition as described above) substituted with amino, methylamino,ethylamino, dimethylamino, diethylamino, pyrrolidinyl, piperidyl ormorpholinyl. Examples thereof include methylcarbamoyl, ethylcarbamoyl,propylcarbamoyl, butylcarbamoyl, pentylcarbamoyl, hexylcarbamoyl,dimethylcarbamoyl, diethylcarbamoyl, ethylmethylcarbamoyl,methylpropylcarbamoyl, butylmethylcarbamoyl, methylpentylcarbamoyl,hexylmethylcarbamoyl, aminoethylcarbamoyl, aminopropylcarbamoyl,(aminoethyl)methylcarbamoyl, bis(aminoethyl)carbamoyl, and the like.Preferred example thereof include methylcarbamoyl, ethylcarbamoyl,dimethylcarbamoyl, and the like.

In the formula (I), it is more preferable that R¹ and R² be identicallylinear or branched alkyl, alkenyl or alkynyl having 12 to 24 carbonatoms. Further preferably, R¹ and R² are both linear or branched alkylor alkenyl having 12 to 24 carbon atoms.

More preferably, L¹ and L² are identically —O—, —CO—O— or —O—CO—.

When at least one of L¹ and L² is —O—, it is more preferable that the R¹and R² attached to —O— are, the same or different, dodecyl, tetradecyl,hexadecyl, octadecyl, icosyl, docosyl, tetracosyl, (Z)-tetradec-9-enyl,(Z)-hexadec-9-enyl, (Z)-octadec-6-enyl, (Z)-octadec-9-enyl,(E)-octadec-9-enyl, (Z)-octadec-11-enyl, (9Z,12Z)-octadec-9,12-dienyl,(9Z,12Z,15Z)-octadec-9,12,15-trienyl, (Z)-icos-11-enyl,(11Z,14Z)-icos-11,14-dienyl, 3,7,11-trimethyldodeca-2,6,10-trienyl or3,7,11,15-tetramethylhexadec-2-enyl, or that the R¹ and R² are combinedtogether to form dialkylmethylene or dialkenylmethylene. Furtherpreferably, R¹ and R² are tetradecyl, hexadecyl, octadecyl,(Z)-hexadec-9-enyl, (Z)-octadec-6-enyl, (Z)-octadec-9-enyl or(9Z,12Z)-octadec-9,12-dienyl, respectively, or are combined together toform di(tetradecyl)methylene, di(hexadecyl)methylene,di(octadecyl)methylene, di((Z)-hexadec-9-enyl)methylene,di((Z)-octadec-6-enyl)methylene, di((Z)-octadec-9-enyl)methylene ordi((9Z,12Z)-octadec-9,12-dienyl)methylene. In all of the case, it iseven more preferable that R¹ and R² are the same or are combinedtogether to form dialkylmethylene, dialkenylmethylene ordialkynylmethylene having the same alkyl, alkenyl or alkynyl moieties.

When at least one of L¹ and L² is O—CO—, it is more preferable that theR¹ and R² attached to —O—CO— are, the same or different, dodecyl,tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, tetracosyl,(Z)-tetradec-9-enyl, (Z)-hexadec-9-enyl, (Z)-octadec-6-enyl,(Z)-octadec-9-enyl, (E)-octadec-9-enyl, (Z)-octadec-11-enyl,(9Z,12Z)-octadec-9,12-dienyl, (9Z,12Z,15Z)-octadec-9,12,15-trienyl,(Z)-icos-11-enyl, (11Z,14Z)-icos-11,14-dienyl,3,7,11-trimethyldodeca-2,6,10-trienyl or3,7,11,15-tetramethylhexadec-2-enyl. Further preferably, R¹ and R² aretetradecyl, hexadecyl, octadecyl, (Z)-hexadec-9-enyl,(Z)-octadec-6-enyl, (Z)-octadec-9-enyl or (9Z,12Z)-octadec-9,12-dienyl,respectively. In all of the case, it is even more preferable that R¹ andR² are the same.

When at least one of L¹ and L² is —CO—O—, it is more preferable that theR¹ and R² attached to —CO—O— are, the same or different, tridecyl,pentadecyl, heptadecyl, nonadecyl, heneicosyl, tricosyl,(Z)-tridec-8-enyl, (Z)-pentadec-8-enyl, (Z)-heptadec-5-enyl,(Z)-heptadec-8-enyl, (E)-heptadec-8-enyl, (Z)-heptadec-10-enyl,(8Z,11Z)-heptadec-8,11-dienyl, (8Z,11Z,14Z)-octadec-8,11,14-trienyl,(Z)-nonadec-10-enyl, (10Z,13Z)-nonadec-10,13-dienyl,(11Z,14Z)-icos-11,14-dienyl, 2,6,10-trimethylundec-1,5,9-trienyl or2,6,10,14-tetramethylpentadec-1-enyl, or are combined together to formdialkylmethylene, dialkenylmethylene, dialkynylmethylene oralkylalkenylmethylene. It is more preferable that R¹ and R² aretridecyl, pentadecyl, heptadecyl, (Z)-pentadec-8-enyl,(Z)-heptadec-5-enyl, (Z)-heptadec-8-enyl or(8Z,11Z)-heptadec-8,11-dienyl, respectively. In all of the case, it iseven more preferable that R¹ and R² are the same or are combinedtogether to form dialkylmethylene, dialkenylmethylene ordialkynylmethylene having the same alkyl, alkenyl or alkynyl moieties.

It is more preferable that a and b are 0 or 1 at the same time.

When a and b are 1 at the same time, it is preferable that X¹ and X² arecombined together to form a single bond or alkylene.

Further preferably, X¹ and X² are combined together to form a singlebond or alkylene. When X¹ and X² are combined together to form a singlebond or alkylene, R³ is preferably a hydrogen atom, methyl,pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having 1 to 6carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to3 substituent(s), which is(are), the same or different, amino,monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidylor morpholinyl. More preferably, R³ is a hydrogen atom, methyl, or alkylhaving 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atomssubstituted with 1 to 3 substituent(s), which is(are), the same ordifferent, amino, hydroxy or carbamoyl. Most preferably, R³ is ahydrogen atom, methyl, 2,3-dihydroxypropyl, 3-hydroxypropyl,aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1,3-diaminopropyl,1,4-diaminobutyl, 1,5-diaminopentyl, 3-aminopropyl, 4-aminobutyl,5-aminopentyl, 2-carbamoylethyl, or the like. Further, when X¹ and X²are combined together to form a single bond, L³ is —CO— or —CO—O—,preferably —CO— in one of the preferred modes of the present invention.In this case, R³ is more preferably aminomethyl, 1,2-diaminoethyl,2-aminoethyl, 1,3-diaminopropyl, 1,4-diaminobutyl, 1,5-diaminopentyl,3-aminopropyl, 4-aminobutyl, 5-aminopentyl, or the like, most preferably1,2-diaminoethyl, 1,3-diaminopropyl, 1,4-diaminobutyl or1,5-diaminopentyl.

Further, when X¹ and X² are combined together to form a single bond, aand b are, the same or different, 1 to 3, preferably 1 in one of thepreferred modes of the present invention. In this case, L¹ and L² areidentically —CO—O— or —O—CO—, preferably —CO—O—, and R³ is methyl in oneof the more preferred modes of the present invention. In this case, itis more preferable that R¹ and R² are, the same or different,(Z)-heptadec-8-enyl or (8Z,11Z)-heptadec-8,11-dienyl. Most preferably,R¹ and R² are identically (Z)-heptadec-8-enyl or(8Z,11Z)-heptadec-8,11-dienyl.

Further, when X¹ and X² are combined together to form a single bond, X³is alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbonatoms, preferably methyl in one of the more preferred modes of thepresent invention. In this case, it is more preferable that L¹ and L²are identically —CO—O— or —O—CO—, most preferably —CO—O—.

When X¹ and X² are hydrogen atoms, it is more preferable that R³ is ahydrogen atom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl,or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbonatoms substituted with 1 to 3 substituent(s), which is(are), the same ordifferent, amino, monoalkylamino, dialkylamino, trialkylammonio,hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl,pyrrolidinyl, piperidyl or morpholinyl. Further preferably, R³ is ahydrogen atom, methyl, or alkyl having 1 to 6 carbon atoms or alkenylhaving 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), whichis(are), the same or different, amino, hydroxy or carbamoyl. Mostpreferably, R³ is a hydrogen atom, methyl, 2,3-dihydroxypropyl,3-hydroxypropyl, aminomethyl, 1,2-diaminoethyl, 2-aminoethyl,1-amino-2-hydroxyethyl, 1,3-diaminopropyl, 1,4-diaminobutyl,1,5-diaminopentyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl,2-carbamoylethyl, or the like.

Preferably, L³ is a single bond. When L³ is a single bond, L¹ and L² aremore preferably —O—.

Further, when L³ is a single bond, R³ is more preferably a hydrogenatom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkylhaving 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atomssubstituted with 1 to 3 substituent(s), which is(are), the same ordifferent, amino, monoalkylamino, dialkylamino, trialkylammonio,hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl,pyrrolidinyl, piperidyl or morpholinyl. Further preferably, R³ is ahydrogen atom, methyl, hydroxymethyl, 2-hydroxyethyl,2,3-dihydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl,2-hydroxy-3-methoxypropyl, aminomethyl, 2-aminoethyl, 3-aminopropyl,4-aminobutyl, 5-aminopentyl, 2-(N,N-dimethylamino)ethyl,3-(N,N-dimethylamino) propyl, 2-carbamoylethyl,2-dimethylcarbamoylethyl, 1-methylpiperidin-4-yl, or the like. Mostpreferably, R³ is a hydrogen atom, methyl, 2,3-dihydroxypropyl,3-hydroxypropyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl,5-aminopentyl, 2-carbamoylethyl, or the like. In all of the case, L¹ andL² are more preferably —O—.

In one of the more preferred modes of the present invention, L¹ and L²are identically —CO—O— or —O—CO—, preferably —CO—O— when X³ and Y do notexist, L³ is a single bond, and R³ is a hydrogen atom. In this case, itis more preferable that R¹ and R² are, the same or different,(Z)-heptadec-5-enyl or (Z)-heptadec-8-enyl. Most preferably, R¹ and R²are identically (Z)-heptadec-5-enyl or (Z)-heptadec-8-enyl.

Further, when L³ is —CO— or —CO—O—, it is more preferable that L¹ and L²are identically —CO—O— or —O—CO—, further preferably —CO—O—.

When L³ is —CO— or —CO—O—, it is more preferable that R³ ispyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having 1 to 6carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to3 substituent(s), which is(are), the same or different, amino,monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidylor morpholinyl, wherein at least one of the substituents is amino,monoalkylamino, dialkylamino, trialkylammonio, pyrrolidinyl, piperidylor morpholinyl. Further preferably, R³ is aminomethyl, 1,2-diaminoethyl,2-aminoethyl, 1,3-diaminopropyl, 3-aminopropyl, 1,4-diaminobutyl,4-aminobutyl, 1,5-diaminopentyl, 5-aminopentyl,(N,N-dimethylamino)methyl, 2-(N,N-dimethylamino)ethyl,3-(N,N-dimethylamino) propyl, 1-amino-2-hydroxyethyl, or the like. Mostpreferably, R³ is 1,2-diaminoethyl, 2-aminoethyl, 1,3-diaminopropyl,3-aminopropyl, 1,4-diaminobutyl, 4-aminobutyl, 1,5-diaminopentyl,5-aminopentyl, or the like.

When L³ is —CO— or —CO—O—, R³ is aminomethyl, 1-hydroxy-2-aminoethyl,2-aminoethyl, 1,3-diaminopropyl, 3-aminopropyl, 1,4-diaminobutyl,4-aminobutyl, 1,5-diaminopentyl or 5-aminopentyl, and L¹ and L² areidentically —O— in one of the more preferred modes of the presentinvention. In this case, it is more preferable that R¹ and R² are, thesame or different, (Z)-octadec-9-enyl or (9Z,12Z)-octadec-9,12-dienyl.Most preferably, R¹ and R² are identically (Z)-octadec-9-enyl or(9Z,12Z)-octadec-9,12-dienyl.

It is more preferable that X³ is absent or is methyl. When X³ is methyl,it is more preferable that R³ is methyl, and that L¹ and L² areidentically —CO—O— or —O—CO—, further preferably —CO—O—.

Production methods of Compound (I) are described below. In the followingproduction methods, in the case where the defined group or groups changeunder the conditions of the production method or are impertinent forcarrying out the production method, the target compound can be producedby adopting common introduction and removal methods of a protectivegroup in synthetic organic chemistry [for example, a method described inProtective Groups in Organic Synthesis, Third Edition, T. W. Greene,John Wiley & Sons Inc. (1999), etc.]. In addition, if desired, the orderof reaction steps such as introduction of a substituent can be altered.

Production Method 1

Among the Compound (I), Compound (Ia) in which L¹ and L² are —O—, L³ isa single bond, and X³ and Y are absent can be produced by the followingmethod.

(In the formula, R¹, R², R³, X¹, X², a and b have the same definitionsas described above, respectively, and Z represents a leaving group suchas a chlorine atom, a bromine atom, an iodine atom,trifluoromethanesulfonyloxy, methanesulfonyloxy, benzenesulfonyloxy, andp-toluenesulfonyloxy.)

Steps 1 and 2

Compound (IIb) can be produced by treating Compound (IIa) and Compound(IIIa) in a solvent in the presence of 1 to 30 equivalents of a base ata temperature between room temperature and 150° C. for 5 minutes to 100hours, followed by isolation. Compound (Ia) can be produced by treatingCompound (IIb) and Compound (IIIb) in a solvent in the presence of 1 to30 equivalents of a base at a temperature between room temperature and150° C. for 5 minutes to 100 hours, followed by isolation.

Examples of the solvent include toluene, diethyl ether, tetrahydrofuran,1,2-dimethoxyethane, dioxane, N,N-dimethylformamide,N-methylpyrrolidone, dimethylsulfoxide, and the like. These may be usedeither alone or as a mixture.

Examples of the base include sodium hydride, sodium hydroxide, potassiumhydroxide, sodium tert-butoxide, potassium tert-butoxide, and the like.

Compound (IIa) can be obtained as a commercially available product or byknown methods (for example, Chemical & Pharmaceutical Bulletin (Chem.Pharm. Bull.), 1991, Vol. 39, p. 2219, and WO2006/10036) or a method inconformity thereof, or by using the methods described in ReferenceExamples.

Compound (IIIa) and Compound (IIIb) can be obtained as commerciallyavailable products or by known methods (for example, Dai 5-han, JikkenKagaku Kouza (5th edition. Courses in Experimental Chemistry) 13,“Synthesis of Organic Compounds I”, 5th Ed., p. 374, Maruzen (2005)), ora method in conformity thereof.

Compound (Ia) having the identical R¹ and R² can be obtained by using 2equivalents or more of the Compound (IIIa) in step 1.

Production Method

Among the Compound (I), Compound (Ib) in which L¹ and L² are —CO—O—, L³is a single bond, and X³ and Y are absent can be produced by thefollowing method.

(In the formula, R¹, R², R³, X¹, X², a and b have the same definitionsas described above, respectively.)

Steps 3 and 4

Compound (IIc) can be produced by treating Compound (IIa) and Compound(IVa) in a solvent in the presence of 1 to 30 equivalents of acondensing agent at a temperature between −20° C. and 150° C. for 5minutes to 100 hours, followed by isolation. Compound (Ib) can beproduced by treating Compound (IIc) and Compound (IVb) in a solvent inthe presence of 1 to 30 equivalents of a condensing agent at atemperature between −20° C. and 150° C. for 5 minutes to 100 hours,followed by isolation. In steps 3 and 4, 0.01 to 30 equivalents of anadditive and/or 1 equivalent to large excess amounts of a base may beadded to promote the reactions.

Examples of the solvent include dichloromethane, chloroform,1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether,tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide,N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, and thelike. These may be used either alone or as a mixture.

Examples of the condensing agent include 1,3-dicyclohexylcarbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.hydrochloride,carbonyldiimidazole, benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate, (benzotriazol-1-yloxy)tripyrrolizinophosphoniumhexafluorophosphate,O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, 2-chloro-1-methylpyridinium iodide, and the like.

Examples of the additive include 1-hydroxybenzotriazole,4-dimethylaminopyridine, and the like.

Examples of the base include potassium acetate, sodium bicarbonate,potassium carbonate, potassium hydroxide, sodium hydroxide, sodiummethoxide, potassium tert-butoxide, triethylamine,diisopropylethylamine, N-methylmorpholine, pyridine,1,8-diazabicyclo[5.4.0]-7-undecene, and the like.

Compound (IVa) and Compound (IVb) can be obtained as commerciallyavailable products or by known methods (for example, Dai 5-han, JikkenKagaku Kouza (5th edition, Courses in Experimental Chemistry) 16,Synthesis of Organic Compounds IV 5th Ed., p. 1, Maruzen (2005)), or amethod in conformity thereof.

Compound (Ib) having the identical R¹ and R² can be obtained by using 2equivalents or more of Compound (IVa) in step 3.

Production Method 3

Among the Compound (I), Compound (Ic) in which L¹ and L² are —O—CO—, L³is a single bond, and X³ and Y are absent can be produced by thefollowing method.

(In the formula, R¹, R², R³, X¹, X², a and b have the same definitionsas described above, respectively.)

Steps 5 and 6

Compound (IIe) can be produced by treating Compound (IId) and Compound(Va) in a solvent in the presence of 1 to 30 equivalents of a condensingagent at a temperature between −20° C. and 150° C. for 5 minutes to 100hours, followed by isolation. Compound (Ic) can be produced by treatingCompound (IIe) and Compound (Vb) in a solvent in the presence of 1 to 30equivalents of a condensing agent at a temperature between −20° C. and150° C. for 5 minutes to 100 hours, followed by isolation. In steps 5and 6, 0.01 to 30 equivalents of an additive and/or 1 equivalent tolarge excess amounts of a base may be added to promote the reactions.

The same solvents, condensing agents, additives, and bases used inproduction method 2 may be used.

Compound (IId) can be obtained as a commercially available product or byknown methods (for example, Dai 5-han, Jikken Kagaku Kouza (5th edition,Courses in Experimental Chemistry) 16, Synthesis of Organic CompoundsIV, 5th Ed., p. 1, Maruzen (2005)), or a method in conformity thereof.

Compound (Va) and Compound (Vb) can be obtained as commerciallyavailable products or by known methods (for example, Dai 5-han, JikkenKagaku Kouza (5th edition, Courses in Experimental Chemistry) 14,Synthesis of Organic Compounds II, 5th Ed., p. 1, Maruzen (2005)), or amethod in conformity thereof.

Compound (Ic) having the identical R¹ and R² may be obtained by using 2equivalents or more of Compound (Va) in step 5.

Production Method 4

Among the Compound (I), Compound (Id) in which L³ is a single bond, R³is a hydrogen atom and, X³ and Y are absent can be produced by thefollowing method.

(In the formula, R¹, R², L¹, L², X¹, X², a and b have the samedefinitions as described above, respectively.)

Step 7

Compound (Id) can be produced by treating Compound (VI) and1-chloroethyl chloroformate in an inert solvent at a temperature between−20° C. and 230° C. for 5 minutes to 100 hours, and then at atemperature between −20° C. and 230° C. for 5 minutes to 100 hours afteradding 1 to large excess amounts of an alcohol.

Examples of the inert solvent include dichloromethane, chloroform,1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether,tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide,N,N-dimethylacetamide, N-methylpyrrolidone, and the like. These may beused either alone or as a mixture.

Examples of the alcohol include methanol, ethanol, 1-propanol,2-propanol, and the like. These may be used either alone or as amixture.

Compound (VI) can be obtained by using a modified method of productionmethod 1, 2 or 3.

Production Method 5

Among the Compound (I), Compound (Ie) can be produced by the followingmethod. In Compound (Ie), L³ is a single bond, R³ is —CHR^(A)R^(B)(R^(A) and R^(B) are, the same or different, hydrogen atoms, alkylhaving 1 to 5 carbon atoms, alkenyl having 3 to 5 carbon atoms,pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl,piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, or alkyl having 1 to 5carbon atoms or alkenyl having 3 to 5 carbon atoms substituted with 1 to3 substituent(s), which is(are), the same or different, amino,monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidylor morpholinyl, or R^(A) and R^(B) are combined together with theadjacent carbon atom thereto to form pyrrolidin-3-yl, piperidin-3-yl orpiperidin-4-yl. The sum of the carbon atoms in the alkyl, the alkylmoiety of the substituted alkyl, alkenyl, and the alkenyl moiety of thesubstituted alkenyl in R^(A) and R^(B) is 1 to 5 except when R^(A) andR^(B) are both hydrogen atoms. When either of R^(A) and R^(B) ispyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl,piperidin-4-yl, morpholin-2-yl or morpholin-3-yl, the other is ahydrogen atom, alkyl having 1 to 5 carbon atoms, alkenyl having 3 to 5carbon atoms, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl,piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, or alkylhaving 1 to 5 carbon atoms or alkenyl having 3 to 5 carbon atomssubstituted with 1 or 2 substituent(s), which is(are), the same ordifferent, amino, monoalkylamino, dialkylamino, trialkylammonio,hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl,pyrrolidinyl, piperidyl or morpholinyl. The total number of thesubstituents is 2 or 3 when R^(A) and R^(B) are substituted alkyl orsubstituted alkenyl), and X³ and Y do not exist.

(In the formula, R¹, R², R^(A), R^(B), L¹, L², X¹, X², a and b have thesame definitions as described above, respectively.)

Step 8

Compound (Ie) can be produced by reacting Compound (Id) with preferably1 to 10 equivalents of Compound (VII) in a solvent at a temperaturebetween −20° C. and 150° C. for 5 minutes to 72 hours in the presence ofpreferably 1 to large excess amounts of a reducing agent, and, ifnecessary, preferably 1 to 10 equivalents of an acid.

Examples of the solvent include methanol, ethanol, dichloromethane,chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile,diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane,N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone,water, and the like. These may be used either alone or as a mixture.

Examples of the reducing agent include sodium triacetoxyborohydride,sodium cyanoborohydride, and the like.

Examples of the acid include hydrochloric acid, acetic acid, and thelike.

Compound (VII) can be obtained as a commercially available product or byknown methods (for example, Dai 5-han, Jikken Kagaku Kouza (5th edition,Courses in Experimental Chemistry) 15, Synthesis of Organic CompoundsIII, 5th Ed., p. 1, Maruzen (2005), and Dai 5-han, Jikken Kagaku Kouza15, Synthesis of Organic Compounds III, 5th Ed., p. 153, Maruzen(2005)), or a method in conformity thereof.

Production Method 6

Among the Compound (I), Compound (If) in which L³ is a single bond, andX³ and Y are absent can be produced by the following method.

(In the formula, R¹, R², R³, L¹, L², X¹, X², a, b, and Z have the samedefinitions as described above, respectively.)

Step 9

Compound (If) can be produced by reacting Compound (Id) with Compound(VIII) without solvent or in a solvent at a temperature between −20° C.and 150° C. for 5 minutes to 72 hours, in the presence of preferably 1to 10 equivalents of an additive, and/or preferably 1 to 10 equivalentsof a base, if necessary.

Examples of the solvent include methanol, ethanol, dichloromethane,chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile,diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane,N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone,pyridine, water, and the like. These may be used either alone or as amixture.

Examples of the base include potassium carbonate, potassium hydroxide,sodium hydroxide, sodium methoxide, potassium tert-butoxide,triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine,1,8-diazabicyclo[5.4.0]-7-undecene (DBU), and the like.

Examples of the additive include sodium iodide, potassium iodide,tetra-n-butylammonium iodide, and the like.

Compound (VIII) can be obtained as a commercially available product orby known methods (for example, Dai 5-han, Jikken Kagaku Kouza (5thedition, Courses in Experimental Chemistry) 13, Synthesis of OrganicCompounds I, 5th Ed., p. 374, Maruzen (2005)), or a method in conformitythereof.

Production Method 7

Among the Compound (I), Compound (Ig) in which L³ is —CO—, and X³ and Yare absent can be produced by the following method.

(In the formula, R¹, R², R³, L¹, L², X¹, X², a and b have the samedefinitions as described above, respectively.)

Step 10

Compound (Ig) can be produced by treating Compound (Id) and Compound(IX) in a solvent at a temperature between −20° C. and 150° C. for 5minutes to 100 hours in the presence of 1 equivalent to large excessamounts of a condensing agent. If necessary, preferably 0.01 to 10equivalents of an additive, and/or preferably 1 to large excess amountsof a base may be added to promote the reaction.

Examples of the solvent include methanol, ethanol, dichloromethane,chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile,diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane,N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone,dimethylsulfoxide, water, and the like. These may be used either aloneor as a mixture.

The same condensing agents, additives, and bases used in productionmethod 2 may be used.

Compound (IX) can be obtained as a commercially available product or byknown methods (for example, Dai 5-han, Jikken Kagaku Kouza (5th edition,Courses in Experimental Chemistry) 16, Synthesis of Organic CompoundsIV, 5th Ed., p. 1, Maruzen (2005)), or a method in conformity thereof.

Production Method 8

Among the Compound (I), Compound (Ih) in which L³ is —CO—O—, and X³ andY are absent can be produced by the following methods.

(In the formula, R¹, R², R³, L¹, L², X¹, X², a and b have the samedefinitions as described above, respectively.)

Step 11

Compound (Ih) can be produced by reacting Compound (Id) with Compound(X) without solvent or in a solvent at a temperature between −20° C. and150° C. for 5 minutes to 72 hours, in the presence of preferably 1 to 10equivalents of an additive, and/or preferably 1 to 10 equivalents of abase, if necessary.

The same solvents and additives used in production method 2 may be used.

Examples of the base include triethylamine, diisopropylethylamine,N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, andthe like.

Compound (X) can be obtained as a commercially available product or byknown methods (for example, Journal of American Chemical Society (J. Am.Chem. Soc.), 1981, Vol. 103, p. 4194-4199), or a method in conformitythereof.

Production Method 9

Among the Compound (I), Compound (Ii) can be produced by the followingmethod. In Compound (Ii), L³ is a single bond, R³ is—CH₂—C(OH)R^(C)R^(D) (R^(C) and R^(D) are, the same or different,hydrogen atoms, alkyl having 1 to 4 carbon atoms, alkenyl having 3 to 4carbon atoms, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl,piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, or alkylhaving 1 to 4 carbon atoms or alkenyl having 3 to 4 carbon atomssubstituted with 1 or 2 substituent(s), which is(are), the same ordifferent, amino, monoalkylamino, dialkylamino, trialkylammonio,hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl,pyrrolidinyl, piperidyl or morpholinyl. The sum of the carbon atoms inthe alkyl, the alkyl moiety of the substituted alkyl, alkenyl, and thealkenyl moiety of the substituted alkenyl in R^(C) and R^(D) is 1 to 4except when R^(C) and R^(D) are both hydrogen atoms. When either ofR^(C) and R^(D) is pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl,piperidin-3-yl, piperidin-4-yl, morpholin-2-yl or morpholin-3-yl, theother is a hydrogen atom, alkyl having 1 to 4 carbon atoms, alkenylhaving 3 to 4 carbon atoms, pyrrolidin-2-yl, pyrrolidin-3-yl,piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl,morpholin-3-yl, or alkyl having 1 to 4 carbon atoms or alkenyl having 3to 4 carbon atoms substituted with a substituent(s), which is(are)amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidylor morpholinyl. The total number of the substituents is 2 when R^(C) andR^(D) are substituted alkyl or substituted alkenyl), and X³ and Y do notexist.

(In the formula, R¹, R², R^(C), R^(D), L¹, L², X¹, X², a and b have thesame definitions as described above, respectively.)

Step 12

Compound (Ii) can be produced by treating Compound (Id) and Compound(XI) in the absence or presence of a solvent at a temperature between 0°C. and 230° C. for 5 minutes to 100 hours.

Examples of the solvent include methanol, ethanol, 1-propanol,dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate,acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane,dioxane, N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpyrrolidone, and dimethyl sulfoxide. These solvents are usedsolely or in admixture.

Compound (XI) can be obtained as a commercially available product or bya known method (for example, Dai 5-han, Jikken Kagaku Kouza (5thedition, Courses in Experimental Chemistry) 17, “Synthesis of OrganicCompounds V”, 5th edition, p. 186, Maruzen (2005)) or a method inconformity therewith.

Production Method 10

Among the Compound (I), Compound (Ij) in which L³ is a single bond, X³is alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbonatoms, and Y is a pharmaceutically acceptable anion can be produced bythe following method.

(In the formula, R¹, R², R³, L¹, L², X¹, X², X³, Y, a, b, and Z have thesame definitions as described above, respectively.)

Steps 13 and 14

Compound (Ij-A) can be produced by treating Compound (If) and Compound(XII) in a solvent or without solvent at a temperature between 0° C. and230° C. for 5 minutes to 100 hours. Compound (Ij) can be produced bytreating Compound (Ij-A) with Y-type anion-exchange resin.

Examples of the solvent include methanol, ethanol, dichloromethane,chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile,diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane,N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone,pyridine, and the like. These may be used either alone or as a mixture.

Compound (XII) can be obtained as a commercially available product or byknown methods (for example, Dai 5-han, Jikken Kagaku Kouza (5th edition,Courses in Experimental Chemistry) 13, Synthesis of Organic Compounds I,5th Ed., p. 374, Maruzen (2005)), or a method in conformity thereof.

When Z and Y are identical, Compound (Ij) may be produced by omittingstep 14.

Production Method 11

Among the Compound (I), Compound (Id) in which L³ is a single bond, R³is a hydrogen atom, and X³ and Y are absent also can be produced by thefollowing method.

(In the formula, R¹, R², L¹, L², X¹, X², a and b have the samedefinitions as described above, respectively.)

Step 15

Compound (Id) can be produced by reacting Compound (XIII) withoutsolvent or in a solvent at a temperature between −20° C. and 150° C. for5 minutes to 100 hours in the presence of preferably 1 to large excessamounts of an acid.

The same solvents used in production method 2 may be used.

Examples of the acid include trifluoroacetic acid, hydrochloric acid,and sulfuric acid.

Compound (XIII) can be obtained by using a modified method of productionmethod 1, 2 or 3.

Conversion of the functional groups contained in R¹, R² or R³ inCompound (I) can be performed by known methods [for example, methodsdescribed in Comprehensive Organic Transformations, 2nd edition, R. C.Larock, Vch Verlagsgesellschaft Mbh (1999), etc.], or a method inconformity thereof.

The intermediates and the target compounds in the foregoing productionmethods can be isolated and purified by using the common separation andpurification techniques used in organic synthesis chemistry, including,for example, filtration, extraction, washing, drying, concentration,recrystallization, various chromatography techniques, and the like. Theintermediates may be fed to the subsequent reactions withoutpurification.

In the Compound (I), a hydrogen ion may coordinate to a lone pair on thenitrogen atom in the structure, and the nitrogen atom may form a salttogether with a pharmaceutically acceptable anion (having the samedefinition as described above). The Compound (I) encompass compounds inwhich a hydrogen ion coordinates to a lone pair on the nitrogen atom.Note that, in the present invention, the absence of X³ encompasses thecase where a hydrogen ion is coordinated.

Compound (I) may exist as stereoisomers (such as geometrical isomers andoptical isomers), tautomers, and the like. Compound (I) encompass all ofpossible isomers and mixtures thereof, inclusive of stereoisomers andtautomers.

A part of or all of the atoms in the Compound (I) may be replaced withcorresponding isotope atoms. The Compound (I) encompass compounds inwhich a part of or all of the atoms thereof are replaced with suchisotope atoms. For example, a part of or all of the hydrogen atoms inthe Compound (I) may be hydrogen atoms having an atomic weight of 2(deuterium atoms).

The compounds in which a part of or all of the atoms in the Compound (I)are replaced with corresponding isotope atoms can be produced by usingmethods similar to the foregoing production methods, using commerciallyavailable building blocks. Further, the compounds in which a part of orall of the hydrogen atoms in the Compound (I) are replaced withdeuterium atoms can be synthesized by using various methods, including,for example, (1) a method in which a carboxylic acid or the like isdeuterated using deuterium peroxide under a basic condition (see U.S.Pat. No. 3,849,458), (2) a method in which an alcohol, a carboxylicacid, or the like is deuterated using an iridium complex as a catalystand using heavy water as a deuterium source (see J. Am. Chem. Soc., Vol.124, No. 10, 2092 (2002)), (3) a method in which a fatty acid isdeuterated using palladium-carbon as a catalyst and using only adeuterium gas as a deuterium source (see LIPIDS, Vol. 9, No. 11, 913(1974)), (4) a method in which acrylic acid, methyl acrylate,methacrylic acid, methyl methacrylate, or the like is deuterated using ametal such as platinum, palladium, rhodium, ruthenium, and iridium as acatalyst and using heavy water or heavy water and a deuterium gas as adeuterium source (see Japanese Published Examined Patent Application No.19536/1993, and Japanese Published Unexamined Patent Application No.277648/1986 and No. 275241/1986), and (5) a method in which acrylicacid, methyl methacrylate, or the like is deuterated using a catalystsuch as palladium, nickel, copper, and copper chromite and using heavywater as a deuterium source (see Japanese Published Unexamined PatentApplication No. 198638/1988), and the like.

Specific examples of the Compound (I) obtained in the present inventionare shown in Tables 1 to 17. It should be noted, however, that thecompounds of the present invention are not limited to these.

TABLE 1 Compound No. Structure 1

2

3

4

5

6

7

8

9

10

TABLE 2 Compound No. Structure 11

12

13

14

15

16

17

18

19

20

TABLE 3 Compound No. Structure 21

22

23

24

25

26

27

28

TABLE 4 Compound No. Structure 29

30

31

32

33

34

35

36

37

TABLE 5 Compound No. Structure 38

39

40

41

42

43

44

45

46

47

TABLE 6 Compound No. Structure 48

49

50

51

52

53

54

55

56

TABLE 7 Compound No. Structure 57

58

59

60

61

62

63

64

TABLE 8 Compound No. Structure 65

66

67

68

69

70

71

72

TABLE 9 Compound No. Structure 73

74

75

76

77

78

79

80

TABLE 10 Compound No. Structure 81

82

83

84

85

86

87

TABLE 11 Compound No. Structure 88

89

90

91

92

93

94

TABLE 12 Compound No. Structure 95

96

97

98

99

100

TABLE 13 Compound No. Structure 101

102

103

104

105

106

107

108

109

TABLE 14 Compound No. Structure 110

111

112

113

114

115

116

TABLE 15 Compound No. Structure 117

118

119

120

121

122

TABLE 16 Compound No. Structure 123

124

125

126

127

128

129

TABLE 17 Compound No. Structure 130

131

132

133

134

135

136

The nucleic acid which is used in the present invention may be anymolecule so far as it is a molecule obtained through polymerization ofnucleotide and/or a molecule having an equal function to the nucleotide.Examples thereof include RNA that is a polymer of ribonucleotide; DNAthat is a polymer of deoxyribonucleotide; a chimera nucleic acidcomposed of RNA and DNA; and a nucleotide polymer in which at least onenucleotide of these nucleic acids is substituted with a molecule havingan equal function to the nucleotide. In addition, a derivativecontaining at least one polymerized molecule of nucleotide and/or amolecule having an equal function to the nucleotide is also included inthe nucleic acid of the present invention. Incidentally, in the presentinvention, uridine U in RNA and thymine T in DNA shall be deemed to bereplaced with each other.

Examples of the molecule having an equal function to nucleotide includenucleotide derivatives.

The nucleotide derivative may be any molecule so far as it is a moleculeobtained by applying modification to nucleotide. For example, for thepurpose of enhancing the nuclease resistance or achieving stabilizationfrom other decomposing factor as compared with RNA or DNA, increasingthe affinity with the complementary strand nucleic acid, increasing thecellular permeability, or achieving the visualization, moleculesobtained by applying modification to ribonucleotide ordeoxyribonucleotide are suitably used.

Examples of the nucleotide derivative include a sugar moiety modifiednucleotide, a phosphodiester bond modified nucleotide, and a basemodified nucleotide.

The sugar moiety modified nucleotide may be any nucleotide in which apart or the entirety of the chemical structure of the sugar moiety ofnucleotide is modified or substituted with an arbitrary substituent, orsubstituted with an arbitrary atom. Above all, a 2′-modified nucleotideis preferably used.

Examples of the modifying group in the sugar moiety modified nucleotideinclude 2′-cyano, 2′-alkyl, 2′-substituted alkyl, 2′-alkenyl,2′-substituted alkenyl, 2′-halogen, 2′-O-cyano, 2′-O-alkyl,2′-O-substituted alkyl, 2′-O-alkenyl, 2′-O-substituted alkenyl,2′-S-alkyl, 2′-S-substituted alkyl, 2′-S-alkenyl, 2′-S-substitutedalkenyl, 2′-amino, 2′-NH-alkyl, 2′-NH-substituted alkyl, 2′-NH-alkenyl,2′-NH-substituted alkenyl, 2′-SO-alkyl, 2′-SO-substituted alkyl,2′-carboxy, 2′-CO-alkyl, 2′-CO-substituted alkyl, 2′-Se-alkyl,2′-Se-substituted alkyl, 2′-SiH₂-alkyl, 2′-SiH₂-substituted alkyl,2′-ONO₂, 2′-NO₂, 2′-N₃, 2′-amino acid residue (the residue that thehydroxyl group is removed from the carboxylic acid of amino acid), and2′-O-amino acid residue (having the same definition as above), and thelike. In addition, Examples thereof include a peptide nucleic acid (PNA)[Acc. Chem. Res., 32, 624 (1999)], an oxy-peptide nucleic acid (OPNA)[J. Am. Chem. Soc., 123, 4653 (2001)], a peptide ribonucleic acid (PRNA)[J. Am. Chem. Soc., 122, 6900 (2000)]. The ribose with the substitutionby a modifying group at 2′ position in the present invention alsoencompasses bridged nucleic acids (BNAs) of a structure in which themodifying group at 2′ position is bridged to the 4′ carbon atom,specifically, locked nucleic acids (LNAs) in which the oxygen atom at 2′position is bridged to the 4′ carbon atom via methylene, ethylenebridged nucleic acids (ENAs) [Nucleic Acid Research, 32, e175 (2004)],and the like.

The preferred modifying group in the sugar moiety modified nucleotideinclude 2′-cyano, 2′-halogen, 2′-O-cyano, 2′-alkyl, 2′-substitutedalkyl, 2′-O-alkyl, 2′-O-substituted alkyl, 2′-O-alkenyl,2′-O-substituted alkenyl, 2′-Se-alkyl, and 2′-Se-substituted alkyl. Morepreferred examples include 2′-cyano, 2′-fluoro, 2′-chloro, 2′-bromo,2′-trifluoromethyl, 2′-O-methyl, 2′-O-ethyl, 2′-O-isopropyl,2′-O-trifluoromethyl, 2′-O-[2-(methoxy)ethyl], 2′-O-(3-aminopropyl),2′-O-(2-[N,N-dimethyl]aminooxy)ethyl, 2′-O-[3-(N,N-dimethylamino)propyl], 2′-O-[2-[2-(N,N-Dimethylamino) ethoxy]ethyl],2′-O-[2-(methylamino)-2-oxoethyl], 2′-Se-methyl, and the like. Even morepreferred are 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, and the like.2′-O-methyl and 2′-O-ethyl are most preferable.

The preferred range of the modifying group in the sugar moiety modifiednucleotide may be defined based on its size. Modifying groups of a sizecorresponding to the size of fluoro to the size of —O-butyl arepreferable, and modifying groups of a size corresponding to the size of—O-methyl to the size of —O-ethyl are more preferable.

The alkyl in the modifying group of the sugar moiety modified nucleotideis the same as the above-mentioned definition of the alkyl having acarbon number of 1 to 6 in the cationic lipid of the present invention.

The alkenyl in the modifying group of the sugar moiety modifiednucleotide is the same as the above-mentioned definition of the alkenylhaving a carbon number of 3 to 6 in the cationic lipid of the presentinvention.

Examples of the halogen in the modifying group of the sugar moietymodified nucleotide include a fluorine atom, a chlorine atom, a bromineatom, and an iodine atom.

Examples of the amino acid in amino acid residue include aliphatic aminoacids (specifically, glycine, alanine, valine, leucine, isoleucine, andthe like), hydroxy amino acids (specifically, serine, threonine, and thelike), acidic amino acids (specifically, aspartic acid, glutamic acid,and the like), acidic amino acid amides (specifically, asparagine,glutamine, and the like), basic amino acids (specifically, lysine,hydroxylysine, arginine, ornithine, and the like), sulfur-containingamino acids (specifically, cysteine, cystine, methionine, and the like),imino acids (specifically, proline, 4-hydroxy proline, and the like),and the like.

Examples of the substituents of the substituted alkyl and thesubstituted alkenyl in the sugar moiety modified nucleotide includehalogen (having the same definition as above), hydroxy, sulfanyl, amino,oxo, —O-alkyl (the alkyl moiety of —O-alkyl has the same definition asabove), —S-alkyl (the alkyl moiety of —S-alkyl has the same definitionas above), —NH-alkyl (the alkyl moiety of —NH-alkyl has the samedefinition as above), dialkylaminooxy (the two alkyls of thedialkylaminooxy may be the same or different, and have the samedefinition as above), dialkylamino (the two alkyls of the dialkylaminomay be the same or different, and have the same definition as above),dialkylaminoalkyleneoxy (the two alkyls of the dialkylaminoalkyleneoxymay be the same or different, and have the same definition as above; thealkylene means a group wherein the one hydrogen atom is removed fromabove-defined alkyl), and the like, and number of substituent ispreferably 1 to 3.

The phosphodiester bond modified nucleotide may be any nucleotide inwhich a part or the entirety of the chemical structure of thephosphodiester bond of nucleotide is modified or substituted with anarbitrary substituent, or substituted with an arbitrary atom. Examplesthereof include a nucleotide in which the phosphodiester bond issubstituted with a phosphorothioate bond, a nucleotide in which thephosphodiester bond is substituted with a phosphorodithioate bond, anucleotide in which the phosphodiester bond is substituted with analkylphosphonate bond, and a nucleotide in which the phosphodiester bondis substituted with a phosphoroamidate bond.

The base-modified nucleotide may be any nucleotide in which a part orthe entirety of the chemical structure of the base of nucleotide ismodified or substituted with an arbitrary substituent, or substitutedwith an arbitrary atom. Examples thereof include a nucleotide in whichan oxygen atom in the base is substituted with a sulfur atom, anucleotide in which a hydrogen atom is substituted with an alkyl grouphaving a carbon number of 1 to 6, a nucleotide in which a methyl groupis substituted with a hydrogen atom or an alkyl group having a carbonnumber of 2 to 6, and a nucleotide in which an amino group is protectedby a protective group such as an alkyl group having a carbon number of 1to 6 and an alkanoyl group having a carbon number of 1 to 6.

Furthermore, examples of the nucleotide derivative include those inwhich other chemical substance such as a lipid, phospholipid, phenazine,folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine,coumarin, and a pigment is added to nucleotide or a nucleotidederivative in which at least one of the sugar moiety, the phosphodiesterbond, and the base is modified. Specific examples thereof include5′-polyamine added nucleotide derivatives, cholesterol added nucleotidederivatives, steroid added nucleotide derivatives, bile acid addednucleotide derivatives, vitamin added nucleotide derivatives, Cy5 addednucleotide derivatives, Cy3 added nucleotide derivatives, 6-FAM addednucleotide derivatives, and biotin added nucleotide derivatives.

In addition, the nucleotide derivatives may form, together with othernucleotides or nucleotide derivatives within the nucleic acid, acrosslinked structure such as an alkylene structure, a peptidestructure, a nucleotide structure, an ether structure, and an esterstructure, or a structure combined with at least one of thesestructures.

Examples of the nucleic acids used in the present invention includepreferably nucleic acids that suppress the expression of the targetgene, more preferably nucleic acids that have an activity of suppressingthe expression of the target gene by utilizing RNA interference (RNAi).

The target gene used in the present invention is not particularlylimited, as long as it is expressed through mRNA production. Preferredexamples thereof include genes associated with tumor or inflammation,including, for example, genes that encodes proteins such as vascularendothelial growth factors (hereinafter, “VEGF”), vascular endothelialgrowth factor receptors (hereinafter, “VEGFR”), fibroblast growthfactors, fibroblast growth factor receptors, platelet-derived growthfactors, platelet-derived growth factor receptors, liver cell growthfactors, liver cell growth factor receptors, Kruppel-like factors(hereinafter, “KLF”), Ets transcription factors, nuclear factors, andhypoxia-inducible factors. Specific examples thereof include VEGF genes,VEGFR genes, fibroblast growth factor genes, fibroblast growth factorreceptor genes, platelet-derived growth factor genes, platelet-derivedgrowth factor receptor genes, liver cell growth factor genes, liver cellgrowth factor receptor genes, KLF genes, Ets transcription factor genes,nuclear factor genes, hypoxia-inducible factor genes, and the like.

Preferably, the target gene used in the present invention is a gene thatis expressed, for example, in liver, lungs, kidneys or spleen. Examplesthereof include genes associated with tumor or inflammation (such asabove), hepatitis B virus genome, hepatitis C virus genome, and genesthat encode proteins such as apolipoprotein (APO), hydroxymethylglutaryl (HMG) CoA reductase, kexin type 9 serine protease (PCSK9),factor XII, glucagon receptor, glucocorticoid receptor, leukotrienereceptor, thromboxane A2 receptor, histamine HI receptor, carbonicanhydrase, angiotensin converting enzyme, renin, p53, tyrosinephosphatase (PTP), sodium dependent glucose transporter, tumor necrosisfactor, and interleukin, and the like.

The nucleic acid that suppresses the expression of the target gene maybe any of, for example, double-stranded nucleic acids (such as siRNA(short interference RNA), and miRNA (micro RNA)), single-strandednucleic acid (shRNA (short hairpin RNA), antisense nucleic acids,ribozyme, etc), and the like, provided that, for example, the nucleicacid contains a base sequence complementary to a part of the basesequence of the mRNA of the gene (target gene) encoding a protein andthe like, and that the nucleic acid suppresses the expression of thetarget gene. Double-stranded nucleic acids are preferably used.

The nucleic acids that contain a base sequence complementary to a partof the base sequence of the target gene mRNA are also referred to asantisense strand nucleic acids, and the nucleic acids that contain abase sequence complementary to the base sequence of the antisense strandnucleic acid are also referred to as sense strand nucleic acids. Thesense strand nucleic acids are nucleic acids that can form a doublestrand by pairing with antisense strand nucleic acids, including thenucleic acid itself that has a partial base sequence of the target gene.

The double-stranded nucleic acids are nucleic acids that have twostrands forming a double-stranded portion by pairing. Thedouble-stranded portion is a portion where a double strand is formed bythe base pairing of the nucleotides or derivatives thereof forming adouble-stranded nucleic acid. The base pairs forming the double-strandedportion are typically 15 to 27 bps, preferably 15 to 25 bps, morepreferably 15 to 23 bps, further preferably 15 to 21 bps, particularlypreferably 15 to 19 bps.

Preferred for use as the antisense strand nucleic acid of thedouble-stranded portion are nucleic acids that contain a partialsequence of the target gene mRNA, with or without the substitution,deletion, or addition of 1 to 3 bases, preferably 1 to 2 bases, morepreferably 1 base, and that have a target protein expression suppressingactivity. The length of the single-stranded nucleic acid forming adouble-stranded nucleic acid is typically 15 to 30 bases, preferably 15to 29 bases, more preferably 15 to 27 bases, further preferably 15 to 25bases, particularly preferably 17 to 23 bases, most preferably 19 to 21bases.

The nucleic acid in the antisense strand and/or the sense strand forminga double-stranded nucleic acid may have an additional nucleic acid thatdoes not form a double strand, contiguous from the 3′-end or 5′-end ofthe double-stranded portion. Such portions not forming a double strandare also referred to as an extension (overhang).

The extension in such double-stranded nucleic acids has 1 to 4 bases,typically 1 to 3 bases at the 3′-end or 5′-end of at least one of thestrands. Preferably, the extension has 2 bases, more preferably dTdT orUU. The extension may be present on only one of the antisense strand andthe sense strand, or on both of the antisense strand and the sensestrand. However, double-stranded nucleic acids having extensions on boththe antisense strand and the sense strand are preferably used.

It is also possible to use a sequence contiguous from thedouble-stranded portion and partially or completely matches the targetgene mRNA, or a sequence contiguous from the double-stranded portion andmatches the base sequence of the complementary strand of the target genemRNA. Further, the nucleic acid that suppresses the expression of thetarget gene may be, for example, a nucleic acidmolecule that generates adouble-stranded nucleic acid by the activity of a ribonuclease such asDicer (WO2005/089287), or a double-stranded nucleic acid that does nothave a 3′ or 5′ extension.

When the double-stranded nucleic acid is siRNA, the antisense strand hasa base sequence in which at least bases 1 to 17 from the 5′-end to the3′-end are complementary to 17 contiguous bases of the target gene mRNA.Preferably, the antisense strand has a base sequence in which bases 1 to19 from the 5′-end to the 3′-end are complementary to 19 contiguousbases of the target gene mRNA, a base sequence in which bases 1 to 21are complementary to 21 contiguous bases of the target gene mRNA, or abase sequence in which bases 1 to 25 are complementary to 25 contiguousbases of the target gene mRNA.

Further, when the nucleic acid used in the present invention is siRNA,preferably 10 to 70%, more preferably 15 to 60%, further preferably 20to 50% of the sugars in the nucleic acid are riboses substituted with amodifying group at the 2′-position. In the present invention, thesubstitution of the ribose with a modifying group at the 2′-positionmeans the substitution of the hydroxyl group with a modifying group atthe 2′-position. The configuration may be the same as or different fromthe configuration of the ribose hydroxyl group at the 2′-position.Preferably, the configuration is the same as the configuration of theribose hydroxyl group at the 2′-position. The ribose substituted with amodifying group at the 2′-position is included within a 2′-modifiednucleotide from among sugar-modified nucleotides, and the modifyinggroup of the ribose substituted at the 2′-position has the samedefinition as the modifying group of 2′-modified nucleotides.

The nucleic acid used in the present invention includes derivatives inwhich the oxygen atom or the like contained in the phosphate moiety, theester moiety, or the like in the structure of the nucleic acid isreplaced with other atoms, for example, such as a sulfur atom.

In addition, in the sugar binding to the base at the 5′-end of each ofthe antisense strand and the sense strand, the hydroxyl group at the5′-end may be modified with a phosphate group or the foregoing modifyinggroup, or a group which is converted into a phosphate group or theforegoing modifying group by a nucleolytic enzyme or the like in aliving body.

In addition, in the sugar binding to the base at the 3′-end of each ofthe antisense strand and the sense strand, the hydroxyl group at the3′-end may be modified with a phosphate group or the foregoing modifyinggroup, or a group which is converted into a phosphate group or theforegoing modifying group by a nucleolytic enzyme or the like in aliving body.

The single-stranded nucleic acid may be any of nucleic acids thatcontain a sequence complementary to the contiguous 15 to 27 basesequence, preferably 15 to 25 base sequence, more preferably 15 to 23base sequence, further preferably 15 to 21 base sequence, particularlypreferably 15 to 19 base sequence of the target gene, with or withoutthe substitution, deletion, or addition of 1 to 3 bases, preferably 1 to2 bases, more preferably 1 base, and that have a target proteinexpression suppressing activity. Preferred for use is a single-strandednucleic acid having 15 to at most 30 bases, preferably 15 to 29 bases,more preferably 15 to 27 bases, further preferably 15 to 25 bases,particularly preferably 15 to 23 bases.

The single-stranded nucleic acid may be one obtained by connecting theantisense strand and the sense strand of the double-stranded nucleicacid via a spacer sequence. Preferred as the spacer oligonucleotide is asingle-stranded nucleic acid molecule of 6 to 12 bases, with a UUsequence at the 5′-end. Examples of the spacer oligonucleotide contain anucleic acid having the sequence UUCAAGAGA. Either the antisense strandor the sense strand joined by a spacer oligonucleotide may represent the5′-end. Preferably, the single-stranded nucleic acid is asingle-stranded nucleic acid, such as shRNA, that has a stem-loopstructure with a double-stranded portion. Single-stranded nucleic acidssuch as shRNA are typically 50 to 70 bases long.

It is also possible to use nucleic acids at most 70 bases long,preferably at most 50 bases long, further preferably at most 30 baseslong, designed to generate the single-stranded nucleic acid or thedouble-stranded nucleic acid by the activity of ribonuclease or thelike.

In addition, the nucleic acids used in the present invention may beproduced by using known RNA or DNA synthesis techniques, and RNA or DNAmodification techniques. For example, the nucleic acids may bechemically synthesized and obtained from Hokkaido System Science Co.,Ltd.

Examples of the composition in the present invention include acomposition comprising a complex particle of the cationic lipid of thepresent invention and a nucleic acid, a composition comprising a complexparticle of a nucleic acid and the cationic lipid of the presentinvention combined with neutral lipid and/or a polymer, a lipid particleconstituted of the complex particle and a lipid membrane encapsulatingthe complex particle, and the like. Examples of the lipid particleinclude a composition comprising a liposome constituted of the complexparticle and a lipid bilayer encapsulating the complex particle, and thelike. Examples of the complex particle include a complex of a nucleicacid and a membrane constituted of lipid bilayer, a complex of a nucleicacid and a liposome, a complex of a nucleic acid and a micelle, and thelike. Preferred are a complex of a nucleic acid and a micelle, and acomplex of a nucleic acid and a liposome.

The composition in the present invention can be produced by a knownproduction method or a method in conformity therewith and may be acomposition produced by any production method. For example, in theproduction of a liposome as one of the composition, a known preparationmethod of a liposome can be applied. Examples of the known preparationmethod of a liposome include a liposome preparation method by Bangham etal. (see J. Mol. Biol., 1965, Vol. 13, pp. 238-252); an ethanolinjection method (see J. Cell. Biol., 1975, Vol. 66, pp. 621-634); aFrench press method (see FEBS Lett., 1979, Vol. 99, pp. 210-214); afreeze-thawing method (see Arch. Biochem. Biophys., 1981, Vol. 212, pp.186-194); a reverse phase evaporation method (see Proc. Natl. Acad. Sci.USA, 1978, Vol. 75, pp. 4194-4198); and a pH gradient method (see, forexample, Japanese Patents Nos. 2572554 and 2659136, etc.). As a solutionwhich disperses the liposome in the production of liposome, for example,water, an acid, an alkali, a variety of buffer solution, a saline, anamino acid infusion, and the like can be used. In addition, in theproduction of a liposome, it is also possible to add an antioxidant, forexample, citric acid, ascorbic acid, cysteine,ethylenediaminetetraacetic acid (EDTA), etc., an isotonic agent, forexample, glycerin, glucose, sodium chloride, etc., or the like. Inaddition, the liposome can also be produced by dissolving a lipid or thelike in an organic solvent, for example, ethanol, etc., distilling offthe solvent, adding a saline or the like, and stirring and shaking themixture, thereby forming a liposome.

In addition, the composition of the present invention can be produced byvarious methods. As an example, the cationic lipid of the presentinvention is dissolved in chloroform in advance, and a nucleic acidaqueous solution and methanol are added. These are mixed to form acationic lipid/nucleic acid complex. Then, the chloroform layer isremoved, and a water-in-oil (W/O) emulsion is formed by addition of apolyethylene glycolated phospholipid, a neutral lipid, and water. Themixture is then treated by using a reverse phase evaporation method (seeJP-T-2002-508765; the term “JP-T” as used herein means a publishedJapanese translation of a PCT patent application). In another method, anucleic acid is dissolved in an acidic electrolytic aqueous solution,and lipid is added (in ethanol) to lower the ethanol concentration to 20v/v % and form the nucleic acid-encapsulating liposome. After sizingfiltration, excess amounts of ethanol are removed by dialysis. Thenucleic acid adhering to the liposome surface is then removed by furtherdialysis at an increased sample pH (see JP-T-2002-501511, and Biochimicaet Biophysica Acta, 2001, Vol. 1510, p. 152-166).

The production methods described in, for example, WO2002/28367 andWO2006/080118 can be used to produce the compositions of the presentinvention, specifically the liposome constituted of complex particle ofthe cationic lipid of the present invention and a nucleic acid, or thecomplex particle of a nucleic acid and the cationic lipid of the presentinvention combined with neutral lipid and/or a polymer, and a lipidbilayer encapsulating the complex particle.

The neutral lipid may be any lipid including a simple lipid, a complexlipid, and a derived lipid. Examples thereof include a phospholipid, aglyceroglycolipid, a sphingoglycolipid, a sphingoid, and a sterol.However, it should not be construed that the present invention islimited thereto.

Examples of the phospholipid in the neutral lipid include natural orsynthetic phospholipids such as phosphatidylcholines (specifically,soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC),distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine(DPPC), palmitoyloleoyl phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), etc.),phosphatidylethanolamines (specifically, distearoylphosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine(DPPE), dioleoyl phosphatidylethanolamine (DOPE), dimyristoylphosphoethanolamine (DMPE), 16-O-monomethyl PE, 16-O-dimethyl PE,18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE),1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), etc.),glycerophospholipids (specifically, phosphatidylserine, phosphatidicacid, phosphatidylglycerol, phosphatidylinositol, palmitoyloleoylphosphatidylglycerol (POPG), lysophosphatidylcholine, etc.),sphingophospholipids (specifically, sphingomyelin, ceramidephosphoethanolamine, ceramide phosphoglycerol, ceramidephosphoglycerophosphate, etc.), a glycerophosphono lipid, asphingophosphonolipid, natural lecithins (specifically, egg yolklecithin, soybean lecithin, etc.), and hydrogenated phospholipids(specifically, hydrogenated soybean phosphatidylcholine, etc.).

Examples of the glyceroglycolipid in the neutral lipid includesulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyldiglyceride, galactosyl diglyceride, and glycosyl diglyceride.

Examples of the sphingoglycolipid in the neutral lipid includegalactosyl cerebroside, lactosyl cerebroside, and ganglioside.

Examples of the sphingoid in the neutral lipid include sphingan,icosasphingan, sphingosine, and a derivative thereof. Examples of thederivative include those in which —NH₂ of sphingan, icosasphingan,sphingosine, or the like is replaced with —NHCO(CH₂)_(x)CH₃ (in theformula, x is an integer of 0 to 18, with 6, 12 or 18 being preferable).

Examples of the sterol in the neutral lipid include cholesterol,dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol,brassicasterol, ergocasterol, fucosterol, and3β-[N—(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol).

The polymer may be one or more micelles selected from, for example,protein, albumin, dextran, polyfect, chitosan, dextran sulfate; andpolymers, for example, such as poly-L-lysine, polyethyleneimine,polyaspartic acid, a copolymer of styrene and maleic acid, a copolymerof isopropylacrylamide and acrylpyrrolidone, polyethylene glycol(PEG)-modified dendrimer, polylactic acid, polylactic acid polyglycolicacid, and polyethylene glycolated polylactic acid, and a salt thereof.

Here, the salt of the polymer includes, for example, a metal salt, anammonium salt, an acid addition salt, an organic amine addition salt, anamino acid addition salt, and the like. Examples of the metal saltinclude alkali metal salts such as a lithium salt, a sodium salt and apotassium salt; alkaline earth metal salts such as a magnesium salt anda calcium salt; an aluminum salt; a zinc salt, and the like. Examples ofthe ammonium salt include salts of ammonium, tetramethylammonium, andthe like. Examples of the acid addition salt include inorganates such asa hydrochloride, a sulfate, a nitrate, and a phosphate, and organatessuch as an acetate, a maleate, a fumarate, and a citrate. Examples ofthe organic amine addition salt include addition salts of morpholine,piperidine, and the like, and examples of the amino acid addition saltinclude addition salts of glycine, phenylalanine, aspartic acid,glutamic acid, lysine, and the like.

Further, the composition of the present invention may comprise, forexample, a lipid conjugate or a fatty acid conjugate of at least onesubstance selected from sugar, peptide, nucleic acid, and water-solublepolymer. The composition may also comprise a surfactant or the like. Alipid conjugate or a fatty acid conjugate of at least one substanceselected from sugar, peptide, nucleic acid, and water-soluble polymer,or a surfactant may be comprised in the composite particle, or may beadded external to the composite particle.

The lipid conjugate or fatty acid conjugate of at least one substanceselected from sugar, peptide, nucleic acid, and water-soluble polymer,or the surfactant is preferably a glycolipid, or a lipid conjugate or afatty acid conjugate of a water-soluble polymer, more preferably a lipidconjugate or a fatty acid conjugate of a water-soluble polymer.Preferably, the lipid conjugate or fatty acid conjugate of at least onesubstance selected from sugar, peptide, nucleic acid, and water-solublepolymer, or the surfactant is a substance having dual properties inwhich a part of the molecule has the property to bind to the otherconstituent components of the composition through, for example,hydrophobic affinity, electrostatic interaction, and the like, whereasother parts of the molecule have the property to bind to the solventused for the production of the composition, through, for example,hydrophilic affinity, electrostatic interaction, and the like.

Examples of the lipid conjugate or fatty acid conjugate of sugar,peptide or nucleic acid include products formed by means of binding ofsugars (such as sucrose, sorbitol, lactose, etc), peptides (such ascasein-derived peptides, egg white-derived peptides, soybean-derivedpeptides, glutathione, etc) or nucleic acids (such as DNA, RNA,plasmids, siRNA ODN, etc) with the neutral lipids as exemplified abovein the definition of the composition or the cationic lipids of thepresent invention, or with fatty acids (such as stearic acid, palmiticacid, myristic acid, lauric acid, etc).

Examples of the lipid conjugate or fatty acid conjugate of sugar includethe glyceroglycolipids, the sphingoglycolipids, and the like asexemplified above in the definition of the composition.

Examples of the lipid conjugate or fatty acid conjugate of water-solublepolymer include products formed by means of binding of, for example,polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol,polyacrylic acid, polyacrylamide, oligosaccharide, dextrin,water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin,chitosan, polyvinylpyrrolidone, polyaspartamide, poly-L-lysine, mannan,pullulan, oligoglycerol, etc, and derivatives thereof with the neutrallipids as exemplified above in the definition of the composition, thecationic lipids of the present invention, or fatty acids (such asstearic acid, palmitic acid, myristic acid, lauric acid, etc). Morepreferred examples thereof include lipid conjugates or fatty acidconjugates of polyethylene glycol derivatives, polyglycerin derivatives,and the like. Further preferred examples thereof include lipidconjugates or fatty acid conjugates of polyethylene glycol derivatives.

Examples of the lipid conjugate or fatty acid conjugate of apolyethylene glycol derivative include a polyethylene glycolated lipid(specifically, polyethylene glycol-phosphatidylethanolamines (morespecifically,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG-DSPE),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG-DMPE), etc.)), polyoxyethylene hydrogenated castoroil 60, CREMOPHOR EL, and the like), a polyethylene glycol sorbitanfatty acid ester (specifically, polyoxyethylene sorbitan monooleate,etc.), and a polyethylene glycol fatty acid ester; preferred examplesthereof include a polyethylene glycolated lipid.

Examples of the lipid conjugate or the fatty acid conjugate of apolyglycerol derivative include a polyglycerolated lipid (specifically,polyglycerol phosphatidyl ethanolamine and the like), a polyglycerolfatty acid ester and the like, and more preferred examples include apolyglycerolated lipid.

Examples of the surfactant include polyoxyethylene sorbitan monooleates(specifically, Polysorbate 80, and the like), polyoxyethylenepolyoxypropylene glycols (specifically, Pluronic F68, and the like),sorbitan fatty acid esters (specifically, sorbitan monolaurate, sorbitanmonooleate, and the like), polyoxyethylene derivatives (specifically,polyoxyethylene hydrogenated castor oil 60, polyoxyethylene laurylalcohol, and the like), glycerin fatty acid esters, and polyethyleneglycolalkyl ethers. Preferred examples thereof include polyoxyethylenepolyoxypropylene glycols, glycerin fatty acid esters, polyethyleneglycolalkyl ethers, and the like.

When the composition of the present invention is a liposome, thecomposition of the liposomemaybe subjectedto anysurfacemodificationwith, for example, apolymer, a polyoxyethylenederivative, and the like. [see D. D. Lasic, F. Martin], StealthLiposomes, CRC Press Inc., US, 1995, p. 93-102]. Examples of polymersusable for the surface modification include dextran, pullulan, mannan,amylopectin, hydroxyethyl starch, and the like. Examples of thepolyoxyethylene derivatives include Polysorbate 80, Pluronic F68,polyoxyethylene hydrogenated castor oil 60, polyoxyethylene laurylalcohol, PEG-DSPE, and the like. The surface modification of thecomposition such as the liposome enables the composition to comprise alipid conjugate or a fatty acid conjugate of at least one substanceselected from sugar, peptide, nucleic acid, and water-soluble polymer,or a surfactant.

The average particle diameter of the composition in the presentinvention may be freely selected as desired. Preferably, the averageparticle diameter of the liposome is adjusted as follows. Examples of amethod for adjusting the average particle diameter include an extrusionmethod, a method in which a large multilamellar liposome vesicle (MLV)and like is mechanically pulverized (specifically, by usingManton-gaulin, a microfluidizer or the like) (see Emulsion andNanosuspensions for the Formulation of Poorly Soluble Drugs, edited byR. H. Muller, S. Benita and B. Bohm, Scientific Publishers, Stuttgart,Germany, pp. 267-294, 1998), and the like.

A complex as a combination of two or more selected from, for example, alipid assembly, a liposome, a polymer micelle, and the like used as thecomposition may be produced simply by mixing the lipid, polymer, and thelike, for example, in water. Other step such as a granulating step and asterilizing step may be added, as desired. The complex may be producedin various solvents, for example, acetone, ether, and the like.

As for the size of the composition in the present invention, an averageparticle diameter is preferably about 10 nm to 1,000 nm, more preferablyabout 30 nm to 300 nm, and still more preferably about 50 nm to 200 nm.

By administering the composition in the present invention to a mammaliancell, the nucleic acid in the composition in the present invention canbe introduced into the cell.

A method for administering the composition in the present invention to amammalian cell in vitro may be carried out according to the proceduresof known transfection capable of being carried out in vitro.

A method for administering the composition of the present invention to amammalian cell in vivo may be carried out according to the procedures ofknown transfection that can be performed in vivo. For example, by theintravenous administration of the composition of the present inventionto mammals including humans, the composition is delivered to, forexample, an organ or a site involving cancer or inflammation, and thenucleic acid in the composition of the present invention can beintroduced into the cells at these organs or sites. The organs or sitesinvolving cancer or inflammation are not particularly limited. Examplesthereof include stomach, large intestine, liver, lungs, spleen,pancreas, kidneys, bladder, skin, blood vessel, and eye ball. Inaddition, by the intravenous administration of the composition of thepresent invention to mammals including humans, the composition can bedelivered to, for example, blood vessel, liver, lungs, spleen, and/orkidneys, and the nucleic acid in the composition of the presentinvention can be introduced into the cells at these organs or sites. Theliver, lung, spleen, and/or kidney cells may be any of normal cells,cells associated with cancer or inflammation, and cells associated withother diseases.

When the nucleic acid in the composition in the present invention is anucleic acid having an activity of suppressing the expression of thetarget gene by utilizing RNA interference (RNAi), nucleic acids such asRNA that suppress the expression of the gene can be introduced tomammalian cells in vivo, and expression of genes can be suppressed. Theadministration target is preferably human.

In addition, when the target gene of composition in the presentinvention is, for example, a gene associated with tumor or inflammation,the composition of the present invention can be used as a therapeuticagent or a preventive agent for cancer or inflammatory disease,preferably a therapeutic agent or a preventive agent for solid cancer orfor inflammation in blood vessels or in the vicinity of blood vessels.Specifically, when the target gene of the composition of the presentinvention is, for example, a gene associated with angiogenesis, thecomposition of the present invention can suppress the proliferation,angiogenesis, or the like in the vascular smooth muscle, and can thus beused as a therapeutic agent or a preventive agent for cancer orinflammatory disease that involves, for example, proliferation orangiogenesis in the vascular smooth muscle.

Specifically, the present invention also provides a cancer orinflammatory disease therapeutic method that includes administering thecomposition of the present invention to a mammal. The administrationtarget is preferably human, more preferably humans having cancer orinflammatory disease.

Further, the composition of the present invention also can be used as atool for acquiring a POC (proof of concept) in an in vivo screeningsystem concerning the cancer or inflammatory disease therapeutic orpreventive agent.

The composition of the present invention also can be used as apreparation for, for example, stabilizing the nucleic acid in biogenicsubstances (for example, blood, digestive tract, and the like) such asblood components, reducing side effects, or increasing drug accumulationin tissues or organs containing the expression site of the target gene.

When the composition of the present invention is used as a medicament,specifically a therapeutic agent or a preventive agent for cancer,inflammatory disease, or the like, it is desirable to use anadministration route that is most effective for the treatment. Theadministration route may be parenteral or oral, including buccaladministration, airway administration, rectal administration,subcutaneous administration, intramuscular administration, intravenousadministration, and the like. Intravenous administration andintramuscular administration are preferable, and intravenousadministration is more preferable.

The dose may vary depending upon factors such as the conditions and theage of a subject, and the administration route. For example, theadministration may be made in a daily dose of, for example, about 0.1 μgto 1,000 mg in terms of the nucleic acid.

As a preparation suitable for the intravenous administration orintramuscular administration, for example, an injection can beexemplified, and it is also possible to use a dispersion liquid of thecomposition prepared by the foregoing method as it is in the form of,for example, an injection or the like. However, it can also be usedafter removing the solvent from the dispersion liquid by, for example,filtration, centrifugation, or the like, or after lyophilizing thedispersion liquid or the dispersion liquid supplemented with anexcipient such as mannitol, lactose, trehalose, maltose, and glycine.

In the case of an injection, it is preferable that an injection isprepared by mixing, for example, water, an acid, an alkali, a variety ofbuffer solution, a saline, an amino acid infusion, or the like with theforegoing dispersion liquid of the composition or the foregoingcomposition obtained by removing the solvent or lyophilization. Inaddition, it is also possible to prepare an injection by adding anantioxidant such as citric acid, ascorbic acid, cysteine, and EDTA, anisotonic agent such as glycerin, glucose, and sodium chloride, or thelike. In addition, it can also be cryopreserved by adding acryopreservation agent such as glycerin.

Next, the present invention is specifically described with reference tothe following Examples and Test Examples. However, it should not beconstrued that the present invention is limited to these Examples andTest Examples.

Incidentally, proton nuclear magnetic resonance spectra (¹H NMR) shownin Examples and Referential Examples are those measured at 270 MHz, 300MHz or 400 MHz, and there may be the case where an exchangeable protonis not distinctly observed depending upon the compound and measuringconditions. Incidentally, the expression for multiplicity of a signal isa usually used expression. The term “br” indicates an apparently broadsignal.

Reference Example 1(3R,4R)-1-Benzyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(compound VI-1)

A toluene (70 mL) solution of (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 3.50 g, 18.1 mmol) was slowly added to a toluene (100mL) suspension of sodium hydride (oily, 60%, 5.80 g, 145 mmol) whilebeing stirred. A toluene (30 mL) solution of(9Z,12Z)-octadec-9,12-dienyl methanesulfonate (Nu-Chek Prep., Inc.; 15.6g, 45.3 mmol) was then dropped on the mixture. The resulting mixture wasstirred overnight under heat and reflux. After cooling the mixture toroom temperature, the reaction was stopped with a saturated ammoniumchloride aqueous solution. After adding saturated brine, the mixture wasextracted twice with ethyl acetate. The organic layers were combined,dried over anhydrous magnesium sulfate, and concentrated under reducedpressure. The residue was purified by silica gel column chromatography(methanol/chloroform=0/100 to 2/98) to give compound VI-1 (6.96 g,55.7%).

ESI-MS m/z: 691 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.9 Hz, 6H),1.26-1.38 (m, 30H), 1.52-1.62 (m, 6H), 2.05 (q, J=6.3 Hz, 8H), 2.50 (dd,J=9.9, 4.3 Hz, 2H), 2.77 (t, J=5.8 Hz, 4H), 2.85 (dd, J=9.6, 5.9 Hz,2H), 3.37-3.45 (m, 4H), 3.52-3.66 (m, 2H), 3.83 (t, J=4.6 Hz, 2H),5.28-5.43 (m, 8H), 7.23-7.33 (m, 5H).

Reference Example 2 (3R,4R)-1-Benzylpyrrolidine-3,4-diyldi((9Z,12Z)-octadec-9,12-dienoate) (compound VI-2)

(3R,4R)-1-Benzylpyrrolidine-3,4-diol (Diverchim S.A.; 350 mg, 1.81 mmol)was dissolved in dichloromethane (18 mL). After adding linoleic acid(Aldrich; 1.24 mL, 3.98 mmol), dicyclohexylcarbodiimide (KokusanChemical Co., Ltd.; 860 mg, 4.17 mmol), and 4-dimethylaminopyridine(Tokyo Chemical Industry Co., Ltd.; 55.3 mg, 0.453 mmol), the mixturewas stirred overnight at room temperature. After adding hexane (18 mL),the reaction mixture was filtered, and concentrated under reducedpressure. The resulting residue was purified by silica gel columnchromatography (hexane/chloroform=40/60 to 20/80) to give compound VI-2(1.21 g, 93.0%).

ESI-MS m/z: 719 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.30-1.40 (m, 28H), 1.55-1.64 (m, 4H), 2.05 (q, J=6.6 Hz, 8H), 2.30 (t,J=7.5 Hz, 4H), 2.50 (dd, J=10.3, 4.0 Hz, 2H), 2.77 (t, J=6.1 Hz, 4H),3.06 (dd, J=10.3, 6.1 Hz, 2H), 3.62 (q, J=13.8 Hz, 2H), 5.12 (dd, J=5.3,4.0 Hz, 2H), 5.28-5.43 (m, 8H), 7.23-7.34 (m, 5H).

Reference Example 3(3R,4R)-1-Benzyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(Compound VI-3)

Compound VI-3 (398 mg, 40.7%) was obtained in the same manner as that inReference Example 1, by using (3R,4S)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 274 mg, 1.42 mmol) and (9Z,12Z)-octadec-9,12-dienylmethanesulfonate (Nu-Chek Prep, Inc; 1.22 g, 3.54 mmol).

ESI-MS m/z: 691 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.6 Hz, 6H),1.29-1.40 (m, 30H), 1.56 (dd, J=13.0, 7.1 Hz, 6H), 2.05 (q, J=6.6 Hz,8H), 2.46 (dd, J=9.5, 6.0 Hz, 2H), 2.77 (t, J=6.0 Hz, 4H), 3.08 (dd,J=9.5, 6.0 Hz, 2H), 3.37-3.53 (m, 4H), 3.63 (s, 2H), 3.85-3.92 (m, 2H),5.28-5.43 (m, 8H), 7.20-7.30 (m, 5H).

Reference Example 4(3R,4R)-1-Benzyl-3,4-bis((Z)-octadec-9-enyloxy)pyrrolidine (CompoundVI-4)

Compound VI-4 (507 mg, 56.4%) was obtained in the same manner as that inReference Example 1, by using (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 250 mg, 1.29 mmol) and (Z)-octadec-9-enylmethanesulfonate (Nu-Chek Prep, Inc; 1.79 g, 5.17 mmol).

ESI-MS m/z: 695 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.36 (m, 44H), 1.53-1.58 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.50 (dd,J=9.9, 4.7 Hz, 2H), 2.85 (dd, J=9.9, 6.1 Hz, 2H), 3.34-3.47 (m, 4H),3.59 (q, J=12.6 Hz, 2H), 3.83 (t, J=4.7 Hz, 2H), 5.29-5.40 (m, 4H),7.23-7.32 (m, 5H).

Reference Example 5 (3R,4R)-1-Benzyl-3,4-bis(tetradecyloxy)pyrrolidine(compound VI-5)

(3R,4R)-1-Benzylpyrrolidine-3,4-diol (Diverchim S.A.; 150 mg, 0.776mmol) was dissolved in dimethylsulfoxide (4 mL). After adding potassiumhydroxide (348 mg, 6.21 mmol), the solution was stirred at 100° C. for15 minutes. The reaction solution was further stirred at 100° C. for 4hours after adding

a dimethylsulfoxide (4 mL) solution of tetradecyl methanesulfonate(Nu-Chek Prep., Inc.; 568 mg, 1.94 mmol). The mixture was cooled to roomtemperature, and, after adding water, the aqueous layer was extractedwith ethyl acetate. The organic layer was washed with water and asaturated sodium chloride aqueous solution, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure afterfiltration. The resulting residue was purified by silica gel columnchromatography (chloroform 100%) to give compound VI-5 (449 mg, 98.6%).

ESI-MS m/z: 587 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.25-1.33 (m, 44H), 1.51-1.60 (m, 4H), 2.50 (dd, J=9.9, 4.7 Hz, 2H),2.85 (dd, J=9.9, 6.0 Hz, 2H), 3.35-3.47 (m, 4H), 3.59 (q, J=12.8 Hz,2H), 3.83 (t, J=4.7 Hz, 2H), 7.21-7.33 (m, 5H).

Reference Example 6(3R,4R)-1-Benzyl-3,4-bis((Z)-hexadec-9-enyloxy)pyrrolidine (CompoundVI-6)

Compound VI-6 (231 mg, 48.0%) was obtained in the same manner as that inReference Example 1, by using (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A. 146 mg, 0.753 mmol) and (Z)-hexadec-9-enylmethanesulfonate (Nu-Chek Prep, Inc; 600 mg, 1.88 mmol).

ESI-MS m/z: 639 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.28-1.37 (m, 36H), 1.50-1.60 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.50 (dd,J=9.8, 4.6 Hz, 2H), 2.85 (dd, J=9.8, 5.9 Hz, 2H), 3.34-3.47 (m, 4H),3.59 (q, J=12.6 Hz, 2H), 3.83 (t, J=4.6 Hz, 2H), 5.29-5.40 (m, 4H),7.20-7.34 (m, 5H).

Reference Example 7(3R,4R)-1-Benzyl-3,4-bis((Z)-octadec-6-enyloxy)pyrrolidine (CompoundVI-7)

Compound VI-7 (196 mg, 40.7%) was obtained in the same manner as that inReference Example 1, by using (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 134 mg, 0.693 mmol) and (Z)-octadec-6-enylmethanesulfonate (Nu-Chek Prep, Inc; 600 mg, 1.73 mmol).

ESI-MS m/z: 695 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.37 (m, 44H), 1.52-1.61 (m, 4H), 1.97-2.05 (m, 8H), 2.50 (dd,J=9.9, 4.6 Hz, 2H), 2.85 (dd, J=9.9, 5.9 Hz, 2H), 3.34-3.48 (m, 4H),3.59 (q, J=11.8 Hz, 2H), 3.83 (t, J=4.6 Hz, 2H), 5.28-5.41 (m, 4H),7.22-7.34 (m, 5H).

Reference Example 8(3R,4R)-1-Benzyl-3,4-bis((11Z,14Z)-icos-11,14-dienyloxy)pyrrolidine(Compound VI-8)

Compound VI-8 (210 mg, 43.7%) was obtained in the same manner as that inReference Example 1, by using (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 124 mg, 0.644 mmol) and (11Z,14Z)-icos-11,14-dienylmethanesulfonate (Nu-Chek Prep, Inc; 600 mg, 1.61 mmol).

ESI-MS m/z: 747 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.27-1.40 (m, 40H), 1.51-1.60 (m, 4H), 2.05 (q, J=6.5 Hz, 8H), 2.50 (dd,J=10.0, 4.5 Hz, 2H), 2.77 (t, J=6.1 Hz, 4H), 2.85 (dd, J=10.0, 6.1 Hz,2H), 3.35-3.47 (m, 4H), 3.59 (q, J=12.8 Hz, 2H), 3.83 (t, J=4.5 Hz, 2H),5.29-5.43 (m, 8H), 7.22-7.33 (m, 5H).

Reference Example 9 (3R,4R)-1-Benzylpyrrolidine-3,4-diyldi((Z)-octadec-9-enoate) (Compound VI-9)

Compound VI-9 (1.85 g, 98.8%) was obtained in the same manner as that inReference Example 2, by using (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 500 mg, 2.59 mmol) and oleic acid (Tokyo ChemicalIndustry Co., Ltd.; 1.61 g, 5.69 mmol).

ESI-MS m/z: 723 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.27-1.35 (m, 40H), 1.55-1.65 (m, 4H), 2.01 (q, J=5.6 Hz, 8H), 2.30 (t,J=7.4 Hz, 4H), 2.50 (dd, J=10.2, 4.1 Hz, 2H), 3.06 (dd, J=10.2, 6.3 Hz,2H), 3.63 (q, J=12.9 Hz, 2H), 5.12 (dd, J=5.1, 4.1 Hz, 2H), 5.28-5.40(m, 4H), 7.23-7.34 (m, 5H).

Reference Example 10(3S,4S)-1-Benzyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(Compound VI-10)

Compound VI-10 (966 mg, 54.1%) was obtained in the same manner as thatin Reference Example 1, by using(3S,4S)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 500 mg, 2.59 mmol)and (9Z,12Z)-octadec-9,12-dienyl methanesulfonate (Nu-Chek Prep, Inc;2.23 g, 6.47 mmol).

ESI-MS m/z: 691 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.4 Hz, 6H),1.28-1.38 (m, 32H), 1.50-1.60 (m, 4H), 2.04 (q, J=6.6 Hz, 8H), 2.49 (dd,J=10.0, 4.1 Hz, 2H), 2.75-2.88 (m, 6H), 3.34-3.47 (m, 4H), 3.59 (q,J=11.2 Hz, 2H), 3.82 (t, J=4.9 Hz, 2H), 5.27-5.43 (m, 8H), 7.21-7.31 (m,5H).

Reference Example 11 (3R,4R)-1-Benzyl-3,4-bis(hexadecyloxy)pyrrolidine(Compound VI-11)

Compound VI-11 (324 mg, 97.6%) was obtained in the same manner as thatin Reference Example 5, by using (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 100 mg, 0.517 mmol) and hexadecyl methanesulfonate(Nu-Chek Prep, Inc; 415 mg, 1.29 mmol).

ESI-MS m/z: 643 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.25-1.33 (m, 52H), 1.50-1.58 (m, 4H), 2.50 (dd, J=9.9, 4.8 Hz, 2H),2.85 (dd, J=9.9, 6.0 Hz, 2H), 3.35-3.47 (m, 4H), 3.59 (q, J=12.8 Hz,2H), 3.83 (t, J=4.8 Hz, 2H), 7.20-7.33 (m, 5H).

Reference Example 12 (3R,4R)-1-Benzyl-3,4-bis(octadecyloxy)pyrrolidine(Compound VI-12)

Compound VI-12 (319 mg, 88.3%) was obtained in the same manner as thatin Reference Example 5, by using(3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 100 mg, 0.517 mmol)and octadecyl methanesulfonate (Nu-Chek Prep, Inc; 451 mg, 1.29 mmol).

ESI-MS m/z: 699 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.25-1.33 (m, 60H), 1.51-1.59 (m, 4H), 2.50 (dd, J=9.8, 4.5 Hz, 2H),2.85 (dd, J=9.8, 6.2 Hz, 2H), 3.35-3.47 (m, 4H), 3.59 (q, J=12.7 Hz,2H), 3.83 (t, J=4.5 Hz, 2H), 7.21-7.33 (m, 5H).

Reference Example 13(3R,4R)-1-Benzyl-3,4-bis((Z)-tetradec-9-enyloxy)pyrrolidine (CompoundVI-13)

Compound VI-13 (119 mg, 49.5%) was obtained in the same manner as thatin Reference Example 1, by using (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 80.0 mg, 0.414 mmol) and (Z)-tetradec-9-enylmethanesulfonate (Nu-Chek Prep, Inc; 301 mg, 1.04 mmol).

ESI-MS m/z: 583 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=7.0 Hz, 6H),1.28-1.37 (m, 28H), 1.51-1.60 (m, 4H), 1.98-2.05 (m, 8H), 2.50 (dd,J=9.8, 4.6 Hz, 2H), 2.85 (dd, J=9.8, 6.0 Hz, 2H), 3.35-3.47 (m, 4H),3.59 (q, J=12.6 Hz, 2H), 3.83 (t, J=4.6 Hz, 2H), 5.29-5.40 (m, 4H),7.21-7.34 (m, 5H).

Reference Example 14(3R,4R)-1-Benzyl-3,4-bis((Z)-octadec-11-enyloxy)pyrrolidine (CompoundVI-14)

Compound VI-14 (244 mg, 60.8%) was obtained in the same manner as thatin Reference Example 1, by using (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 112 mg, 0.577 mmol) and (Z)-octadec-11-enylmethanesulfonate (Nu-Chek Prep, Inc; 500 mg, 1.44 mmol).

ESI-MS m/z: 695 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.35 (m, 44H), 1.51-1.59 (m, 4H), 2.01 (q, J=6.1 Hz, 8H), 2.50 (dd,J=9.9, 4.6 Hz, 2H), 2.85 (dd, J=9.9, 6.0 Hz, 2H), 3.35-3.47 (m, 4H),3.59 (q, J=12.8 Hz, 2H), 3.83 (t, J=4.6 Hz, 2H), 5.30-5.40 (m, 4H),7.21-7.34 (m, 5H).

Reference Example 15(3R,4R)-1-Benzyl-3,4-bis((Z)-icos-11-enyloxy)pyrrolidine (CompoundVI-15)

Compound VI-15 (251 mg, 62.7%) was obtained in the same manner as thatin Reference Example 1, by using (3R,4R)-1-benzylpyrrolidine-3,4-diol(Diverchim S.A.; 103 mg, 0.534 mmol) and (Z)-icos-11-enylmethanesulfonate (Nu-Chek Prep, Inc; 500 mg, 1.34 mmol).

ESI-MS m/z: 751 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 52H), 1.50-1.60 (m, 4H), 2.01 (q, J=6.0 Hz, 8H), 2.50 (dd,J=9.8, 4.4 Hz, 2H), 2.85 (dd, J=9.8, 6.2 Hz, 2H), 3.35-3.47 (m, 4H),3.59 (q, J=12.8 Hz, 2H), 3.83 (t, J=4.4 Hz, 2H), 5.30-5.40 (m, 4H),7.21-7.34 (m, 5H).

Reference Example 16 (trans-1-Benzylpyrrolidine-3,4-diyl)dimethanol

trans-Diethyl 1-benzylpyrrolidine-3,4-dicarboxylate (830 mg, 2.72 mmol)synthesized by using WO2009/027820 as a reference was dissolved in THF(24 mL). After adding lithium aluminum hydride (206 mg, 5.44 mmol) at 0°C., the solution was stirred at room temperature for 1.3 hours. Thereaction mixture was further stirred at room temperature after addingsodium sulfate decahydrate, chloroform, and Celite. The mixture wasfiltered after adding anhydrous magnesium sulfate, and the filtrate wasconcentrated under reduced pressure. Hexane was added, and the solid wasremoved by filtration to give (trans-1-benzylpyrrolidine-3,4-diyl)dimethanol (565 mg, 93.9%).

ESI-MS m/z: 222 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 2.17-2.28 (m, 2H), 2.35 (dd,J=9.0, 5.1 Hz, 2H), 2.77 (dd, J=9.0, 7.1 Hz, 2H), 3.56-3.68 (m, 6H),7.22-7.34 (m, 5H).

Reference Example 17trans-1-Benzyl-3,4-bis(((Z)-hexadec-9-enyloxy)methyl)pyrrolidine(compound VI-16)

Compound VI-16 (372 mg, 82.3%) was obtained in the same manner as thatin Reference Example 1, by using the(trans-1-benzylpyrrolidine-3,4-diyl)dimethanol (150 mg, 0.678 mmol)obtained in Reference Example 16, and (Z)-hexadec-9-enylmethanesulfonate (Nu-Chek Prep., Inc.; 540 mg, 1.70 mmol).

ESI-MS m/z: 667 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.28-1.35 (m, 36H), 1.49-1.57 (m, 4H), 1.95-2.10 (m, 10H), 2.37 (dd,J=9.2, 5.5 Hz, 2H), 2.67 (dd, J=9.2, 7.0 Hz, 2H), 3.31-3.44 (m, 8H),3.57 (dd, J=18.1, 13.0 Hz, 2H), 5.29-5.40 (m, 4H), 7.19-7.32 (m, 5H).

Reference Example 18 N-Benzyldiethanolamine

Diisopropylethylamine (2.99 mL, 17.1 mmol) and benzyl bromide (1.36 mL,11.4 mmol) were added to a chloroform (46 mL) solution of diethanolamine(1.80 g, 17.1 mmol), and the solution was stirred for 5 hours under heatand reflux. The reaction solution was washed with water, saturatedsodium bicarbonate water, and saturated brine, dried over magnesiumsulfate, and evaporated after filtration. The resulting residue waspurified by silica gel column chromatography (methanol/chloroform=0/100to 12/88) to give V-benzyldiethanolamine (1.77 g, 79.4%).

ESI-MS m/z: 196 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 2.29 (br s, 2H), 2.73 (t,J=5.3 Hz, 4H), 3.63 (t, J=5.3 Hz, 4H), 3.71 (s, 2H), 7.24-7.37 (m, 5H).

Reference Example 19N-Benzyl-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)amine (Compound VI-17)

Compound VI-17 (257 mg, 48.4%) was obtained in the same manner as thatin Reference Example 1, by using N-benzyldiethanolamine (149 mg, 0.763mmol) obtained in Reference Example 18 and (Z)-octadec-9-enylmethanesulfonate (Nu-Chek Prep, Inc; 661 mg, 1.91 mmol).

ESI-MS m/z: 697 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H), 1.27(br s, 44H), 1.50-1.58 (m, 4H), 1.97-2.04 (m, 8H), 2.74 (t, J=6.2 Hz,4H), 3.37 (t, J=6.6 Hz, 4H), 3.50 (t, J=6.2 Hz, 4H), 3.71 (s, 2H),5.29-5.40 (m, 4H), 7.21-7.35 (m, 5H).

Reference Example 20N-Benzyl-N,N-bis(2-((Z)-tetradec-9-enyloxy)ethyl)amine (Compound VI-18)

Compound VI-18 (424 mg, 82.9%) was obtained in the same manner as thatin Reference Example 1, by using N-benzyldiethanolamine (171 mg, 0.876mmol) obtained in Reference Example 18 and (Z)-tetradec-9-enylmethanesulfonate (Nu-Chek Prep, Inc; 636 mg, 2.19 mmol)

ESI-MS m/z: 585 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=7.1 Hz, 6H),1.25-1.35 (m, 28H), 1.50-1.57 (m, 4H), 1.97-2.05 (m, 8H), 2.74 (t, J=6.3Hz, 4H), 3.37 (t, J=6.7 Hz, 4H), 3.50 (t, J=6.3 Hz, 4H), 3.71 (s, 2H),5.29-5.40 (m, 4H), 7.19-7.36 (m, 5H).

Reference Example 21 N-Benzyl-N,N-bis(2-(tetradecyloxy)ethyl)amine(Compound VI-19)

Compound VI-19 (173 mg, 33.6%) was obtained in the same manner as thatin Reference Example 5, by using N-benzyldiethanolamine (171 mg, 0.876mmol) obtained in Reference Example 18 and tetradecyl methanesulfonate(Nu-Chek Prep, Inc; 640 mg, 2.19 mmol).

ESI-MS m/z: 589 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H), 1.25(br s, 44H), 1.50-1.58 (m, 4H), 2.74 (t, J=6.1 Hz, 4H), 3.37 (t, J=6.6Hz, 4H), 3.50 (t, J=6.3 Hz, 4H), 3.71 (s, 2H), 7.21-7.36 (m, 5H).

Reference Example 22 N-Benzyl-N,N-bis(2-(hexadecyloxy)ethyl)amine(Compound VI-20)

Compound VI-20 (411 mg, 72.9%) was obtained in the same manner as thatin Reference Example 5, by using N-benzyldiethanolamine (171 mg, 0.876mmol) obtained in Reference Example 18 and hexadecyl methanesulfonate(Nu-Chek Prep, Inc; 702 mg, 2.19 mmol).

ESI-MS m/z: 645 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H), 1.25(br s, 52H), 1.50-1.58 (m, 4H), 2.74 (t, J=6.3 Hz, 4H), 3.37 (t, J=6.6Hz, 4H), 3.50 (t, J=6.3 Hz, 4H), 3.71 (s, 2H), 7.21-7.36 (m, 5H).

Reference Example 23 N-Benzyl-N,N-bis(2-(octadecyloxy)ethyl)amine(Compound VI-21)

Compound VI-21 (421 mg, 68.7%) was obtained in the same manner as thatin Reference Example 5, by using N-benzyldiethanolamine (171 mg, 0.876mmol) obtained in Reference Example 18 and octadecyl methanesulfonate(Nu-Chek Prep, Inc; 763 mg, 2.19 mmol).

ESI-MS m/z: 701 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H), 1.25(br s, 60H), 1.49-1.58 (m, 4H), 2.74 (t, J=6.3 Hz, 4H), 3.37 (t, J=6.6Hz, 4H), 3.50 (t, J=6.3 Hz, 4H), 3.71 (s, 2H), 7.19-7.35 (m, 5H).

Reference Example 24N-Benzyl-N,N-bis(2-((Z)-hexadec-9-enyloxy)ethyl)amine (Compound VI-22)

Compound VI-22 (739 mg, 81.4%) was obtained in the same manner as thatin Reference Example 1, by using N-benzyldiethanolamine (277 mg, 1.42mmol) obtained in Reference Example 18 and (Z)-hexadec-9-enylmethanesulfonate (Nu-Chek Prep, Inc; 1.13 g, 3.55 mmol)

ESI-MS m/z: 641 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.28-1.35 (m, 36H), 1.49-1.58 (m, 4H), 2.01 (q, J=5.5 Hz, 8H), 2.74 (t,J=6.2 Hz, 4H), 3.37 (t, J=6.6 Hz, 4H), 3.50 (t, J=6.2 Hz, 4H), 3.71 (s,2H), 5.29-5.40 (m, 4H), 7.19-7.35 (m, 5H).

Reference Example 25trans-1-Benzyl-3,4-bis(((Z)-octadec-9-enyloxy)methyl)pyrrolidine(Compound VI-23)

Compound VI-23 (359 mg, 73.4%) was obtained in the same manner as thatin Reference Example 1, by using(trans-1-benzylpyrrolidine-3,4-diyl)dimethanol (150 mg, 0.678 mmol)obtained in Reference Example 16 and (Z)-octadec-9-enyl methanesulfonate(Nu-Chek Prep, Inc; 597 mg, 1.70 mmol).

ESI-MS m/z: 723 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.27-1.36 (m, 44H), 1.48-1.57 (m, 4H), 1.98-2.08 (m, 10H), 2.37 (dd,J=9.0, 5.1 Hz, 2H), 2.67 (dd, J=9.0, 7.2 Hz, 2H), 3.31-3.43 (m, 8H),3.52-3.63 (m, 2H), 5.29-5.40 (m, 4H), 7.21-7.31 (m, 5H).

Reference Example 26trans-1-Benzyl-3,4-bis(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)pyrrolidine(Compound VI-24)

Compound VI-24 (384 mg, 78.9%) was obtained in the same manner as thatin Reference Example 1, by using(trans-1-benzylpyrrolidine-3,4-diyl)dimethanol (150 mg, 0.678 mmol)obtained in Reference Example 16 and (9Z,12Z)-octadec-9,12-dienylmethanesulfonate (Nu-Chek Prep, Inc; 584 mg, 1.70 mmol).

ESI-MS m/z: 719 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.4 Hz, 6H),1.28-1.40 (m, 32H), 1.48-1.57 (m, 4H), 2.05 (q, J=6.6 Hz, 10H), 2.37(dd, J=9.0, 4.9 Hz, 2H), 2.67 (dd, J=9.0, 7.1 Hz, 2H), 2.77 (t, J=5.9Hz, 4H), 3.30-3.43 (m, 8H), 3.51-3.63 (m, 2H), 5.28-5.43 (m, 8H),7.20-7.31 (m, 5H).

Reference Example 27trans-1-3,4-bis(((11Z,14Z)-icos-11,14-dienyloxy)methyl)pyrrolidine(Compound VI-25)

Compound VI-25 (423 mg, 80.6%) was obtained in the same manner as thatin Reference Example 1, by using(trans-1-benzylpyrrolidine-3,4-diyl)dimethanol (150 mg, 0.678 mmol)obtained in Reference Example 16 and (11Z,14Z)-icos-11,14-dienylmethanesulfonate (Nu-Chek Prep, Inc; 631 mg, 1.70 mmol).

ESI-MS m/z: 775 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.27-1.38 (m, 40H), 1.49-1.57 (m, 4H), 2.05 (q, J=6.7 Hz, 10H), 2.37(dd, J=9.2, 5.1 Hz, 2H), 2.67 (dd, J=9.1, 7.1 Hz, 2H), 2.77 (t, J=6.0Hz, 4H), 3.31-3.43 (m, 8H), 3.52-3.62 (m, 2H), 5.29-5.43 (m, 8H),7.21-7.31 (m, 5H).

Reference Example 28trans-1-(tert-Butoxycarbonyl)-3,4-bis(((Z)-octadec-9-enoyloxy)methyl)pyrrolidine(Compound XIII-1)

Compound XIII-1 (280 mg, 54.6%) was obtained in the same manner as thatin Reference Example 2, by usingtrans-3,4-bis(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butylester (156 mg, 0.674 mmol) obtained by using the method described inWO2006/100036 and oleic acid (Tokyo Chemical Industry Co., Ltd.; 419 mg,1.48 mmol).

ESI-MS m/z: 761 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.25-1.46 (m, 36H), 1.46 (s, 9H), 1.46-1.66 (m, 8H), 1.97-2.04 (m, 8H),2.27-2.38 (m, 6H), 3.10-3.23 (m, 2H), 3.53-3.66 (m, 2H), 4.03 (dd,J=10.8, 6.0 Hz, 2H), 4.14 (dd, J=10.8, 6.0 Hz, 2H), 5.28-5.40 (m, 4H).

Reference Example 29trans-1-(tert-Butoxycarbonyl)-3,4-bis(((9Z,12Z)-octadec-9,12-dienoyloxy)methyl)pyrrolidine (Compound XIII-2)

Compound XIII-2 (351 mg, 71.7%) was obtained in the same manner as thatin Reference Example 2, by usingtrans-3,4-bis(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butylester (150 mg, 0.674 mmol) obtained by using the method described inWO2006/100036 and linoleic acid (Aldrich; 400 mg, 1.48 mmol)

ESI-MS m/z: 757 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.21-1.45 (m, 26H), 1.46 (s, 9H), 1.47-1.68 (m, 6H), 2.05 (q, J=6.7 Hz,8H), 2.26-2.38 (m, 6H), 2.77 (t, J=5.9 Hz, 4H), 3.10-3.23 (m, 2H),3.53-3.66 (m, 2H), 4.03 (dd, J=11.0, 6.0 Hz, 2H), 4.14 (dd, J=11.0, 6.0Hz, 2H), 5.28-5.43 (m, 8H).

Example 1 (3R,4R)-3,4-bis((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidine(compound 1)

Compound VI-1 (6.96 g, 10.1 mmol) obtained in Reference Example 1 wasdissolved in 1,2-dichloroethane (100 mL), and stirred at 130° C. for 1hour after adding 1-chloroethyl chloroformate (Tokyo Chemical IndustryCo., Ltd.; 3.30 mL, 30.3 mmol). After adding methanol(100 mL), thereaction solution was further stirred at 130° C. for 1 hour. After beingcooled to room temperature, the solution was concentrated under reducedpressure, and the resulting residue was purified by silica gel columnchromatography (chloroform/methanol=100/0 to 92/8). Fractions comprisingthe compound were collected, washed with a saturated sodium bicarbonateaqueous solution and a saturated sodium chloride aqueous solution, anddried over anhydrous magnesium sulfate. After filtration, the residuewas concentrated under reduced pressure to give compound 1 (5.56 g,92.0%).

ESI-MS m/z: 601 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.9 Hz, 6H),1.29-1.41 (m, 30H), 1.49-1.60 (m, 4H), 1.67 (br s, 3H), 2.05 (q, J=6.5Hz, 8H), 2.75-2.85 (m, 6H), 3.09 (dd, J=12.4, 5.1 Hz, 2H), 3.37-3.49 (m,4H), 3.76 (dd, J=5.0, 3.3 Hz, 2H), 5.28-5.43 (m, 8H).

Example 2 (3R,4R)-Pyrrolidine-3,4-diyldi((9Z,12Z)-octadec-9,12-dienoate) (Compound 2)

Compound 2 (1.20 g, 90.9%) was obtained in the same manner as that inExample 1, by using Compound VI-2 (1.51 g, 2.10 mmol) obtained inReference Example 2.

ESI-MS m/z: 629 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.26-1.41 (m, 29H), 1.56-1.68 (m, 4H), 2.05 (q, J=6.4 Hz, 8H), 2.30 (t,J=7.6 Hz, 4H), 2.77 (t, J=5.8 Hz, 4H), 2.87 (dd, J=13.0, 3.0 Hz, 2H),3.32 (dd, J=13.0, 5.0 Hz, 2H), 5.08 (dd, J=5.0, 3.0 Hz, 2H), 5.28-5.44(m, 8H).

Example 3 (3R,4S)-3,4-bis((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidine(Compound 3)

Compound 3 (245 mg, 81.3%) was obtained in the same manner as that inExample 1, by using Compound VI-3 (346 mg, 0.501 mmol) obtained inReference Example 3.

ESI-MS m/z: 601 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.7 Hz, 6H),1.30-1.40 (m, 30H), 1.54-1.68 (m, 8H), 2.05 (q, J=6.7 Hz, 8H), 2.77 (t,J=5.8 Hz, 4H), 3.00 (d, J=5.0 Hz, 3H), 3.41-3.55 (m, 4H), 3.83 (t, J=3.8Hz, 2H), 5.28-5.43 (m, 8H).

Example 4 (3R,4R)-3,4-bis((Z)-Octadec-9-enyloxy)pyrrolidine (Compound 4)

Compound 4 (333 mg, 84.1%) was obtained in the same manner as that inExample 1, by using Compound VI-4 (455 mg, 0.655 mmol) obtained inReference Example 4.

ESI-MS m/z: 605 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.5 Hz, 6H),1.26-1.35 (m, 38H), 1.50-1.58 (m, 11H), 2.01 (q, J=6.5 Hz, 8H), 2.82(dd, J=12.4, 3.0 Hz, 2H), 3.09 (dd, J=12.4, 5.0 Hz, 2H), 3.43 (td,J=6.5, 1.3 Hz, 4H), 3.76 (dd, J=5.0, 3.0 Hz, 2H), 5.30-5.40 (m, 4H).

Example 5 (3R,4R)-3,4-bis(Tetradecyloxy)pyrrolidine (Compound 5)

Compound 5 (331 mg, 86.1%) was obtained in the same manner as that inExample 1, by using Compound VI-5 (454 mg, 0.775 mmol) obtained inReference Example 5.

ESI-MS m/z: 497 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.34 (m, 41H), 1.50-1.59 (m, 4H), 1.66 (br s, 4H), 2.82 (dd,J=12.6, 3.0 Hz, 2H), 3.09 (dd, J=12.6, 5.0 Hz, 2H), 3.40-3.46 (m, 4H),3.76 (dd, J=5.0, 3.0 Hz, 2H).

Example 6 (3R,4R)-3,4-bis((Z)-Hexadec-9-enyloxy)pyrrolidine (Compound 6)

Compound 6 (160 mg, 89.2%) was obtained in the same manner as that inExample 1, by using Compound VI-6 (208 mg, 0.326 mmol) obtained inReference Example 6.

ESI-MS m/z: 549 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.27-1.36 (m, 34H), 1.50-1.59 (m, 4H), 1.82 (br s, 3H), 2.01 (q, J=6.2Hz, 8H), 2.84 (dd, J=12.5, 3.0 Hz, 2H), 3.10 (dd, J=12.5, 5.0 Hz, 2H),3.43 (t, J=6.8 Hz, 4H), 3.77 (dd, J=5.0, 3.0 Hz, 2H), 5.29-5.40 (m, 4H).

Example 7 (3R,4R)-3,4-bis((Z)-Octadec-6-enyloxy)pyrrolidine (Compound 7)

Compound 7 (123 mg, 82.2%) was obtained in the same manner as that inExample 1, by using Compound VI-7 (171 mg, 0.246 mmol) obtained inReference Example 7.

ESI-MS m/z: 605 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.38 (m, 40H), 1.51-1.61 (m, 4H), 1.64 (s, 5H), 1.97-2.06 (m, 8H),2.82 (dd, J=12.5, 3.3 Hz, 2H), 3.09 (dd, J=12.5, 5.1 Hz, 2H), 3.41-3.46(m, 4H), 3.76 (dd, J=4.6, 3.3 Hz, 2H), 5.29-5.41 (m, 4H).

Example 8 (3R,4R)-3,4-bis((11Z,14Z)-Icos-11,14-dienyloxy)pyrrolidine(Compound 8)

Compound 8 (144 mg, 87.5%) was obtained in the same manner as that inExample 1, by using Compound VI-8 (186 mg, 0.249 mmol) obtained inReference Example 8.

ESI-MS m/z: 657 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.27-1.40 (m, 36H), 1.50-1.59 (m, 4H), 1.64 (s, 5H), 2.05 (q, J=6.6 Hz,8H), 2.75-2.85 (m, 6H), 3.09 (dd, J=12.5, 5.0 Hz, 2H), 3.43 (td, J=6.7,1.3 Hz, 4H), 3.76 (dd, J=5.0, 2.9 Hz, 2H), 5.29-5.43 (m, 8H).

Example 9 (3R,4R)-Pyrrolidine-3,4-diyl di((Z)-octadec-9-enoate)(Compound 9)

Compound 9 (965 mg, 61.6%) was obtained in the same manner as that inExample 1, by using Compound VI-9 (1.79 g, 2.48 mmol) obtained inReference Example 9.

ESI-MS m/z: 633 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.36 (m, 38H), 1.56-1.64 (m, 7H), 2.01 (q, J=5.9 Hz, 8H), 2.30 (t,J=7.6 Hz, 4H), 2.87 (dd, J=13.1, 2.8 Hz, 2H), 3.32 (dd, J=13.1, 5.1 Hz,2H), 5.09 (dd, J=5.1, 2.8 Hz, 2H), 5.28-5.41 (m, 4H).

Example 10(3R,4R)-1-Methyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(compound 10)

Compound 1 (4.00 g, 6.67 mmol) obtained in Example 1 was dissolved in1,2-dichloroethane (50 mL) and methanol (50 mL), and stirred at roomtemperature for 1 hour after adding formaldehyde (4.96 mL, 66.7 mmol)and sodium triacetoxyborohydride (Acros Organics; 7.06 g, 33.3 mmol).The reaction mixture was further stirred at room temperature for 2.5hours after adding sodium triacetoxyborohydride (Acros Organics; 7.06 g,33.3 mmol). The aqueous layer was extracted with ethyl acetate afteradding a saturated sodium bicarbonate aqueous solution to the reactionsolution. The organic layer was washed with a saturated sodium chlorideaqueous solution, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure after filtration. The resultingresidue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 95/5) to give compound 10 (3.99 g, 97.4%).

ESI-MS m/z: 615 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.30-1.41 (m, 30H), 1.52-1.62 (m, 4H), 1.70 (br s, 2H), 2.05 (q, J=6.5Hz, 8H), 2.31 (s, 3H), 2.47 (dd, J=9.9, 4.0 Hz, 2H), 2.75-2.86 (m, 6H),3.36-3.49 (m, 4H), 3.81 (dd, J=5.5, 4.5 Hz, 2H), 5.28-5.44 (m, 8H).

Example 11(3R,4S)-1-Methyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(Compound 11)

Compound 11 (129 mg, 64.6%) was obtained in the same manner as that inExample 10, by using Compound 3 (194 mg, 0.323 mmol) obtained in Example3.

ESI-MS m/z: 615 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.30-1.40 (m, 28H), 1.54-1.62 (m, 4H), 1.76 (br s, 4H), 2.05 (q, J=5.9Hz, 8H), 2.38 (s, 3H), 2.46-2.51 (m, 2H), 2.77 (t, J=5.9 Hz, 4H),3.06-3.11 (m, 2H), 3.39-3.55 (m, 4H), 3.90 (t, J=3.8 Hz, 2H), 5.28-5.43(m, 8H).

Example 12 (3R,4R)-1-Methyl-3,4-bis((Z)-octadec-9-enyloxy)pyrrolidine(Compound 12)

Compound 12 (81.0 mg, 79.4%) was obtained in the same manner as that inExample 10, by using Compound 4 (100 mg, 0.166 mmol) obtained in Example4.

ESI-MS m/z: 619 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.23-1.35 (m, 44H), 1.52-1.61 (m, 4H), 2.01 (q, J=5.8 Hz, 8H), 2.31 (s,3H), 2.46 (dd, J=9.8, 4.4 Hz, 2H), 2.82 (dd, J=9.8, 5.8 Hz, 2H),3.37-3.48 (m, 4H), 3.81 (t, J=4.4 Hz, 2H), 5.30-5.40 (m, 4H).

Example 13 (3R,4R)-1-Methyl-3,4-bis(tetradecyloxy)pyrrolidine (Compound13)

Compound 13 (73.6 mg, 96.8%) was obtained in the same manner as that inExample 10, by using Compound 5 (74.0 mg, 0.149 mmol) obtained inExample 5.

ESI-MS m/z: 511 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.26-1.35 (m, 44H), 1.52-1.61 (m, 4H), 2.31 (s, 3H), 2.47 (dd, J=9.8,4.2 Hz, 2H), 2.83 (dd, J=9.8, 5.5 Hz, 2H), 3.37-3.48 (m, 4H), 3.81 (dd,J=5.5, 4.2 Hz, 2H).

Example 14 (3R,4R)-3,4-bis((Z)-Hexadec-9-enyloxy)-1-methylpyrrolidine(Compound 14)

Compound 14 (107 mg, 97.4%) was obtained in the same manner as that inExample 10, by using Compound 6 (107 mg, 0.195 mmol) obtained in Example6.

ESI-MS m/z: 563 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.38 (m, 34H), 1.52-1.62 (m, 4H), 1.67 (br s, 2H), 2.01 (q, J=6.1Hz, 8H), 2.32 (s, 3H), 2.47 (dd, J=9.8, 4.4 Hz, 2H), 2.83 (dd, J=9.8,5.8 Hz, 2H), 3.36-3.49 (m, 4H), 3.81 (t, J=4.4 Hz, 2H), 5.29-5.41 (m,4H).

Example 15 (3R,4R)-1-Methyl-3,4-bis((Z)-octadec-6-enyloxy)pyrrolidine(Compound 15)

Compound 15 (75.3 mg, 91.8%) was obtained in the same manner as that inExample 10, by using Compound 7 (80.0 mg, 0.132 mmol) obtained inExample 7.

ESI-MS m/z: 619 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.41 (m, 44H), 1.53-1.63 (m, 4H), 1.97-2.06 (m, 8H), 2.31 (s, 3H),2.46 (dd, J=9.6, 4.2 Hz, 2H), 2.82 (dd, J=9.6, 5.6 Hz, 2H), 3.36-3.49(m, 4H), 3.81 (dd, J=5.6, 4.2 Hz, 2H), 5.28-5.41 (m, 4H).

Example 16(3R,4R)-3,4-bis((11Z,14Z)-Icos-11,14-dienyloxy)-1-methylpyrrolidine(Compound 16)

Compound 16 (87.4 mg, 95.0%) was obtained in the same manner as that inExample 10, by using Compound 8 (90.0 mg, 0.137 mmol) obtained inExample 8.

ESI-MS m/z: 671 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.27-1.41 (m, 40H), 1.52-1.61 (m, 4H), 2.05 (q, J=6.5 Hz, 8H), 2.31 (s,3H), 2.46 (dd, J=10.0, 4.5 Hz, 2H), 2.77 (t, J=5.7 Hz, 4H), 2.82 (dd,J=10.0, 5.7 Hz, 2H), 3.36-3.49 (m, 4H), 3.81 (t, J=4.5 Hz, 2H),5.28-5.43 (m, 8H).

Example 17(3R,4R)-1-Ethyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(Compound 17)

Compound 1 (70.0 mg, 0.117 mmol) obtained in Example 1 was dissolved inethanol (2 mL), and stirred at room temperature for 2 days after addingpotassium carbonate (32.2 mg, 0.233 mmol), iodoethane (0.0104 mL, 0.128mmol). The reaction solution was concentrated under reduced pressure.The resulting residue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 96/4) to give compound 17 (33.3 mg,45.4%).

ESI-MS m/z: 629 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.9 Hz, 6H), 1.11(t, J=6.8 Hz, 2H), 1.30-1.38 (m, 29H), 1.55 (br s, 10H), 2.05 (q, J=6.6Hz, 8H), 2.53 (br s, 2H), 2.77 (t, J=5.6 Hz, 4H), 2.88 (br s, 2H), 3.44(t, J=6.6 Hz, 4H), 3.83 (t, J=4.6 Hz, 2H), 5.28-5.44 (m, 8H).

Example 18 (3R,4R)-1-Ethyl-3,4-bis((Z)-octadec-9-enyloxy)pyrrolidine(Compound 18)

Compound 18 (13.4 mg, 32.0%) was obtained in the same manner as that inExample 17, by using Compound 4 (40.0 mg, 0.066 mmol) obtained inExample 4.

ESI-MS m/z: 633 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H), 1.08(t, J=7.1 Hz, 3H), 1.23-1.35 (m, 44H), 1.52-1.59 (m, 4H), 2.01 (q, J=6.2Hz, 8H), 2.37-2.52 (m, 4H), 2.84 (dd, J=9.5, 6.2 Hz, 2H), 3.41-3.45 (m,4H), 3.81 (t, J=4.9 Hz, 2H), 5.29-5.40 (m, 4H).

Example 19(3R,4R)-1,1-Dimethyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidiniumchloride (compound 19)

Iodomethane (1 mL) was added to compound 10 (24.6 mg, 0.0401 mmol)obtained in Example 10, and the mixture was stirred at room temperaturefor 4 hours. The reaction mixture was concentrated under reducedpressure, and the residue was loaded into an anion-exchange resin (Dowex1x-200 chloride type; The Dow Chemical Company; 0.5 mL; prewashed withwater and methanol), and eluted with methanol. The eluate wasconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (methanol/chloroform=0/100 to 25/75) togive compound 19 (24.9 mg, 93.5%).

ESI-MS m/z: 629 M⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H), 1.29 (s,32H), 1.50-1.57 (m, 4H), 1.58 (s, 4H), 2.05 (q, J=6.7 Hz, 8H), 2.77 (t,J=5.9 Hz, 4H), 3.44-3.57 (m, 4H), 3.67 (s, 6H), 3.86 (dd, J=13.4, 3.8Hz, 2H), 4.04-4.13 (m, 4H), 5.29-5.42 (m, 8H).

Example 20(3R,4R)-1,1-Dimethyl-3,4-bis((9Z,12Z)-octadec-9,12-dienoyloxy)pyrrolidinium chloride (compound 20)

Compound 20 (1.21 g, 96.4%) was obtained in the same manner as that inExample 19, by using compound A-3 (1.16 g, 1.81 mmol) obtained inReference Example 30.

ESI-MS m/z: 657 M⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H), 1.29-1.38(m, 26H), 1.57-1.67 (m, 4H), 1.78 (s, 2H), 2.05 (q, J=6.6 Hz, 8H), 2.39(t, J=7.6 Hz, 4H), 2.77 (t, J=5.8 Hz, 4H), 3.78 (s, 6H), 4.15 (dd,J=14.0, 3.0 Hz, 2H), 4.38 (dd, J=14.0, 5.8 Hz, 2H), 5.27-5.46 (m, 10H).

Example 21(3R,4S)-1,1-Dimethyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidiniumchloride (compound 21)

Compound 21 (62.8 mg, 76.6%) was obtained in the same manner as that inExample 19, by using compound 11 (76.0 mg, 0.124 mmol) obtained inExample 11.

ESI-MS m/z: 629 M⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.7 Hz, 6H), 1.23-1.41(m, 30H), 1.52-1.61 (m, 4H), 1.93-2.17 (m, 2H), 2.05 (q, J=6.7 Hz, 8H),2.77 (t, J=5.8 Hz, 4H), 3.41 (s, 3H), 3.47-3.64 (m, 9H), 4.43-4.50 (m,2H), 4.58 (br s, 2H), 5.28-5.44 (m, 8H).

Example 22(3R,4R)-1,1-Dimethyl-3,4-bis((Z)-octadec-9-enyloxy)pyrrolidiniumchloride (compound 22)

Compound 4 (135 mg, 0.223 mmol) obtained in Example 4 was dissolved inmethanol (2 mL), and stirred overnight at room temperature after addingpotassium carbonate (154 mg, 1.12 mmol), and iodomethane (0.699 mL, 11.2mmol). The reaction solution was concentrated under reduced pressure,and the residue was loaded into an anion-exchange resin (Dowex 1x-200chloride type; The Dow Chemical Company; 1 mL; prewashed with water andmethanol), and eluted with methanol. The eluate was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (methanol/chloroform=0/100 to 70/30) to give compound 22(27.8 mg, 18.6%).

ESI-MS m/z: 633 M⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H), 1.26-1.36(m, 40H), 1.50-1.59 (m, 4H), 1.62 (s, 4H), 2.01 (q, J=5.9 Hz, 8H),3.44-3.58 (m, 4H), 3.66 (s, 6H), 3.86 (dd, J=13.2, 4.0 Hz, 2H),4.02-4.13 (m, 4H), 5.29-5.41 (m, 4H).

Example 23 (3R,4R)-1,1-Dimethyl-3,4-bis((Z)-octadec-9-enoyloxy)pyrrolidinium chloride (compound 23)

Compound 23 (442 mg, 95.2%) was obtained in the same manner as that inExample 19, by using compound A-4 (430 mg, 0.666 mmol) obtained inReference Example 31.

ESI-MS m/z: 661 M⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H), 1.26-1.35(m, 38H), 1.58-1.67 (m, 4H), 1.76 (br s, 2H), 2.01 (q, J=5.1 Hz, 8H),2.38 (t, J=7.5 Hz, 4H), 3.78 (s, 6H), 4.15 (dd, J=13.6, 2.6 Hz, 2H),4.37 (dd, J=13.6, 5.7 Hz, 2H), 5.29-5.40 (m, 4H), 5.43-5.46 (m, 2H).

Example 243-((3R,4R)-3,4-bis((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidin-1-yl)propane-1,2-dio1 (compound 24)

Compound 1 (100 mg, 0.167 mmol) obtained in Example 1 was dissolved in1-propanol (1 mL), and irradiated with microwave (300 W, 100° C., 2hours) after adding glycidol (0.111 mL, 1.67 mmol). The reactionsolution was concentrated under reduced pressure after adding water. Theresulting residue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 87/13) to give compound 24 (30.4 mg,27.1%).

ESI-MS m/z: 675 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.9 Hz, 3.0H), 0.93(t, J=7.3 Hz, 3.0H), 1.26-1.38 (m, 22.0H), 1.51-1.67 (m, 8.0H), 2.05 (q,J=6.4 Hz, 8.0H), 2.33-2.42 (m, 1.0H), 2.54 (dd, J=10.2, 4.3 Hz, 1.0H),2.66-2.90 (m, 7.0H), 3.04 (dd, J=9.9, 5.9 Hz, 1.0H), 3.40-3.58 (m,9.5H), 3.63-3.88 (m, 7.5H), 5.28-5.44 (m, 8.0H).

Example 25(3R,4R)-1-(2-(Dimethylamino)acetyl)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine (compound 25)

Compound 1 (100 mg, 0.167 mmol) obtained in Example 1 was dissolved inchloroform (2 mL), and stirred at room temperature for 1.5 hours afteradding N,N-dimethylglycine hydrochloride (Tokyo Chemical Industry Co.;46.5 mg, 0.333 mmol), diisopropylethylamine (0.146 mL, 0.833 mmol), and(benzotriazol-1-yloxy)tripyrrolizinophosphonium hexafluorophosphate(Watanabe Chemical Industries, Ltd.; 217 mg, 0.417 mmol). The aqueouslayer was extracted with chloroform after adding a saturated sodiumbicarbonate aqueous solution to the reaction mixture. The organic layerwas washed with a saturated sodium chloride aqueous solution, dried overanhydrous magnesium sulfate, and concentrated under reduced pressureafter filtration. The resulting residue was purified by silica gelcolumn chromatography (chloroform/methanol=100/0 to 95/5) to givecompound 25 (98.9 mg, 86.8%).

ESI-MS m/z: 686 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.29-1.41 (m, 32H), 1.49-1.57 (m, 4H), 2.05 (q, J=6.5 Hz, 8H), 2.31 (s,6H), 2.77 (t, J=5.8 Hz, 4H), 3.06 (s, 2H), 3.37-3.70 (m, 8H), 3.84-3.91(m, 2H), 5.28-5.43 (m, 8H).

Example 26 (3R,4R)-1-(2-(Dimethylamino)acetyl)pyrrolidine-3,4-diyldi((9Z,12Z)-octadec-9,12-dienoate) (compound 26)

Compound 26 (297 mg, 87.2%) was obtained in the same manner as that inExample 25, by using compound 2 (300 mg, 0.478 mmol) obtained in Example2, and N,N-dimethylglycine hydrochloride (Tokyo Chemical Industry Co.,Ltd.; 133 mg, 0.955 mmol).

ESI-MS m/z: 714 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.29-1.40 (m, 28H), 1.56-1.64 (m, 4H), 2.05 (q, J=6.6 Hz, 8H), 2.27-2.35(m, 10H), 2.77 (t, J=5.7 Hz, 4H), 3.06 (s, 2H), 3.66-3.91 (m, 4H), 5.18(d, J=4.0 Hz, 2H), 5.28-5.43 (m, 8H).

Example 27 (3R,4R)-1-(2-(Dimethylamino)acetyl)pyrrolidine-3,4-diyldi((Z)-octadec-9-enoate) (Compound 27)

Compound 27 (210 mg, 92.5%) was obtained in the same manner as that inExample 25, by using compound 9 (200 mg, 0.316 mmol) obtained in Example9 and N,N-dimethylglycine hydrochloride (Tokyo Chemical Industry Co.,Ltd.; 88 mg, 0.633 mmol).

ESI-MS m/z: 718 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 40H), 1.56-1.64 (m, 4H), 2.01 (q, J=6.2 Hz, 8H), 2.27-2.34(m, 10H), 3.06 (s, 2H), 3.66-3.91 (m, 4H), 5.18 (d, J=3.7 Hz, 2H), 5.34(tt, J=11.2, 4.6 Hz, 4H).

Example 28(3R,4R)-1-(3-(Dimethylamino)propanoyl)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine (Compound 28)

Compound 28 (43.2 mg, 58.8%) was obtained in the same manner as that inExample 25, by using compound 1 (63.0 mg, 0.105 mmol) obtained inExample 1 and 3-(dimethylamino)propionic acid (MATRIX Scientific; 24.6mg, 0.210 mmol).

ESI-MS m/z: 700 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.28-1.38 (m, 32H), 1.48-1.57 (m, 4H), 2.05 (q, J=6.5 Hz, 8H), 2.75-2.85(m, 6H), 2.95 (s, 6H), 3.42-3.53 (m, 8H), 3.59-3.66 (m, 2H), 3.86-3.95(m, 2H), 5.28-5.44 (m, 8H).

Example 29 (3R,4R)-1-(3-(dimethylamino)propanoyl)pyrrolidine-3,4-diyldi((9Z,12Z)-octadec-9,12-dienoate) (Compound 29)

Compound 29 (57.6 mg, 82.9%) was obtained in the same manner as that inExample 25, by using compound 2 (60.0 mg, 0.096 mmol) obtained inExample 2 and 3-(dimethylamino)propionic acid (MATRIX Scientific; 22.4mg, 0.191 mmol).

ESI-MS m/z: 728 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=7.0 Hz, 6H),1.26-1.40 (m, 28H), 1.55-1.63 (m, 4H), 2.05 (q, J=6.7 Hz, 8H), 2.27-2.34(m, 10H), 2.44 (t, J=7.4 Hz, 2H), 2.68 (t, J=7.4 Hz, 2H), 2.77 (t, J=5.7Hz, 4H), 3.55 (d, J=12.1 Hz, 1H), 3.64-3.78 (m, 2H), 3.82 (dd, J=12.1,4.0 Hz, 1H), 5.18 (d, J=4.0 Hz, 2H), 5.28-5.43 (m, 8H).

Example 30 (3R,4R)-1-(3-(Dimethylamino)propanoyl)pyrrolidine-3,4-diyldi((Z)-octadec-9-enoate) (Compound 30)

Compound 30 (209 mg, 90.3%) was obtained in the same manner as that inExample 25, by using compound 9 (200 mg, 0.316 mmol) obtained in Example9 and 3-(dimethylamino)propionic acid (MATRIX Scientific; 74.1 mg, 0.633mmol).

ESI-MS m/z: 732 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 38H), 1.56-1.65 (m, 4H), 1.74 (br s, 2H), 2.01 (q, J=5.5Hz, 8H), 2.28-2.34 (m, 10H), 2.46 (t, J=7.3 Hz, 2H), 2.72 (t, J=7.3 Hz,2H), 3.55 (d, J=12.1 Hz, 1H), 3.67-3.85 (m, 3H), 5.19 (d, J=3.7 Hz, 2H),5.29-5.40 (m, 4H).

Example 31(3R,4R)-1-((S)-2,6-Diaminohexanoyl)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine (compound 31)

Nε-(tert-Butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine(Tokyo Chemical Industry Co., Ltd.; 125 mg, 0.267 mmol), and(benzotriazol-1-yloxy)tripyrrolizinophosphonium hexafluorophosphate(Watanabe Chemical Industries, Ltd.; 146 mg, 0.280 mmol) were dissolvedin chloroform (1 mL), and stirred at room temperature for 1 hour. Achloroform (2 mL) solution of compound 1 (80.0 mg, 0.133 mmol) obtainedin Example 1 was added to the reaction solution, and the mixture wasstirred at room temperature for 7 hours. The reaction mixture wasstirred overnight at room temperature after addingNε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine(Tokyo Chemical Industry Co., Ltd.; 187 mg, 0.400 mmol), and(benzotriazol-1-yloxy)tripyrrolizinophosphonium hexafluorophosphate(Watanabe Chemical Industries, Ltd.; 222 mg, 0.427 mmol). The reactionmixture was further stirred overnight at room temperature, and at 80° C.for 3 hours after addingNε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine(Tokyo Chemical Industry Co., Ltd.; 156 mg, 0.333 mmol), andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (Aldrich; 152 mg, 0.400 mmol). A saturated sodiumbicarbonate aqueous solution was added after cooling the mixture to roomtemperature, and the aqueous layer was extracted with ethyl acetate. Theorganic layer was washed with a saturated sodium chloride aqueoussolution, dried over anhydrous magnesium sulfate, and concentrated underreduced pressure after filtration. The resulting residue was then passedthrough a silica gel pad to give a crude product of(5S)-5-(((9H-fluoren-9-yl)methoxy)carbonylamino)-6-(3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)-6-oxohexylcarbamicacid tert-butyl ester.

The resulting crude product was dissolved in dichloromethane (2 mL), andthe solution was stirred at room temperature for 4 hours after addingtrifluoroacetic acid (0.103 mL, 1.33 mmol). The reaction mixture wasstirred at room temperature for 4.5 hours after adding trifluoroaceticacid (0.205 mL, 2.67 mmol). The aqueous layer was extracted withchloroform after adding a saturated sodium bicarbonate aqueous solutionto the reaction solution. The organic layer was washed with a saturatedsodium chloride aqueous solution, dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure after filtration. Theresulting residue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 87/13) to give(9H-fluoren-9-yl)methyl(2S)-6-amino-1-(3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)-1-oxohexan-2-yl carbamate (66.1 mg, 52.2% in 2 steps).

The resulting(9H-fluoren-9-yl)methyl(2S)-6-amino-1-(3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)-1-oxohexan-2-yl carbamate (65 mg, 0.068 mmol) wasdissolved in tetrahydrofuran (2 mL), and stirred at room temperature for5 hours after adding pyrrolidine (0.5 mL). The reaction mixture wasconcentrated under reduced pressure, and the resulting residue waspurified by silica gel column chromatography (chloroform/methanol=100/0to 60/40) to give compound 31 (24.0 mg, 48.2%).

ESI-MS m/z: 729 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.9 Hz, 6H),1.26-1.38 (m, 32H), 1.50-1.74 (m, 10H), 2.05 (q, J=6.5 Hz, 8H), 2.77 (t,J=5.8 Hz, 4H), 3.01 (br s, 2H), 3.32-3.72 (m, 8H), 3.86 (br s, 2H), 3.93(br s, 1H), 5.28-5.43 (m, 8H).

Example 32N-Methyl-N,N-bis(2-((9Z,12Z)-octadec-9,12-dienyloxy)ethyl)amine(Compound 32)

Compound 32 (68.3 mg, 11.1%) was obtained in the same manner as that inReference Example 1, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 119 mg, 0.999 mmol) and (9Z,12Z)-octadec-9,12-dienylmethanesulfonate (Nu-Chek Prep, Inc; 861 mg, 2.50 mmol).

ESI-MS m/z: 617 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.9 Hz, 6H), 1.29(br s, 32H), 1.50-1.61 (m, 4H), 2.00-2.09 (m, 8H), 2.33 (s, 3H), 2.64(t, J=6.1 Hz, 4H), 2.77 (t, J=5.6 Hz, 4H), 3.41 (t, J=6.8 Hz, 4H), 3.52(t, J=6.1 Hz, 4H), 5.27-5.44 (m, 8H).

Example 33 N-Methyl-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)amine(Compound 33)

Compound 33 (156 mg, 25.2%) was obtained in the same manner as that inReference Example 1, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 119 mg, 0.999 mmol) and (Z)-octadec-9-enylmethanesulfonate (Nu-Chek Prep, Inc; 865 mg, 2.50 mmol).

ESI-MS m/z: 621 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.25-1.34 (m, 44H), 1.51-1.60 (m, 4H), 1.97-2.04 (m, 8H), 2.33 (s, 3H),2.63 (t, J=6.1 Hz, 4H), 3.41 (t, J=6.8 Hz, 4H), 3.52 (t, J=6.1 Hz, 4H),5.28-5.40 (m, 4H).

Example 34 N-Methyl-N,N-bis(2-(tetradecyloxy)ethyl)amine (Compound 34)

Compound 34 (99.3 mg, 0.194 mmol) was obtained in the same manner asthat in Reference Example 1, by using N-methyldiethanolamine (TokyoChemical Industry Co., Ltd.; 119 mg, 0.999 mmol) and tetradecylmethanesulfonate (Nu-Chek Prep, Inc; 731 mg, 2.50 mmol).

ESI-MS m/z: 513 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H), 1.26(br s, 44H), 1.51-1.60 (m, 4H), 2.33 (s, 3H), 2.64 (t, J=6.1 Hz, 4H),3.41 (t, J=6.8 Hz, 4H), 3.52 (t, J=5.9 Hz, 4H).

Example 35 N-Methyl-N,N-bis(2-((Z)-hexadec-9-enyloxy)ethyl)amine(Compound 35)

Compound 35 (199 mg, 50.9%) was obtained in the same manner as that inReference Example 1, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 82.6 mg, 0.693 mmol) and (Z)-hexadec-9-enylmethanesulfonate (Nu-Chek Prep, Inc; 530 mg, 1.66 mmol).

ESI-MS m/z: 565 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H), 1.29(br s, 36H), 1.51-1.56 (m, 4H), 1.97-2.04 (m, 8H), 2.33 (s, 3H), 2.64(t, J=6.1 Hz, 4H), 3.41 (t, J=6.8 Hz, 4H), 3.52 (t, J=6.1 Hz, 4H),5.28-5.40 (m, 4H).

Example 36 N-Methyl-N,N-bis(2-((Z)-octadec-6-enyloxy)ethyl)amine(Compound 36)

Compound 36 (205 mg, 59.4%) was obtained in the same manner as that inReference Example 1, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 66.3 mg, 0.557 mmol) and (Z)-octadec-6-enylmethanesulfonate (Nu-Chek Prep, Inc; 463 mg, 1.34 mmol).

ESI-MS m/z: 621 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.24-1.37 (m, 44H), 1.52-1.63 (m, 4H), 1.97-2.06 (m, 8H), 2.33 (s, 3H),2.64 (t, J=6.1 Hz, 4H), 3.41 (t, J=6.6 Hz, 4H), 3.52 (t, J=6.1 Hz, 4H),5.29-5.40 (m, 4H).

Example 37 N-Methyl-N,N-bis(2-(octadecyloxy)ethyl)amine (Compound 37)

Compound 37 (218 mg, 23.3%) was obtained in the same manner as that inReference Example 5, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 179 mg, 1.50 mmol) and 1-bromooctadecane (TokyoChemical Industry Co., Ltd.; 1.20 g, 3.60 mmol)

ESI-MS m/z: 625 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H), 1.26(s, 60H), 1.51-1.60 (m, 4H), 2.33 (s, 3H), 2.64 (t, J=6.1 Hz, 4H), 3.41(t, J=6.6 Hz, 4H), 3.52 (t, J=6.1 Hz, 4H).

Example 38 (3R,4R)-3,4-bis(Hexadecyloxy)pyrrolidine (Compound 38)

Compound 38 (210 mg, 84.8%) was obtained in the same manner as that inExample 1, by using Compound VI-11 (288 mg, 0.449 mmol) obtained inReference Example 11.

ESI-MS m/z: 553 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.34 (m, 50H), 1.50-1.59 (m, 4H), 1.66-1.68 (m, 3H), 2.82 (dd,J=12.5, 3.0 Hz, 2H), 3.09 (dd, J=12.5, 5.0 Hz, 2H), 3.43 (td, J=6.6, 0.7Hz, 4H), 3.76 (dd, J=5.0, 3.0 Hz, 2H).

Example 39 (3R,4R)-3,4-bis(Octadecyloxy)pyrrolidine (Compound 39)

Compound 39 (209 mg, 82.6%) was obtained in the same manner as that inExample 1, by using Compound VI-12 (290 mg, 0.415 mmol) obtained inReference Example 12.

ESI-MS m/z: 609 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.34 (m, 58H), 1.50-1.59 (m, 4H), 1.64 (br s, 3H), 2.82 (dd,J=12.3, 3.1 Hz, 2H), 3.09 (dd, J=12.3, 5.0 Hz, 2H), 3.43 (t, J=6.6 Hz,4H), 3.77 (dd, J=5.0, 3.1 Hz, 2H).

Example 40 (3R,4R)-3,4-bis((Z)-Tetradec-9-enyloxy)pyrrolidine (Compound40)

Compound 40 (71.4 mg, 84.0%) was obtained in the same manner as that inExample 1, by using Compound VI-13 (100 mg, 0.172 mmol) obtained inReference Example 13.

ESI-MS m/z: 492 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.87-0.92 (m, 6H), 1.29-1.35(m, 26H), 1.50-1.59 (m, 4H), 1.64 (br s, 3H), 2.02 (q, J=5.9 Hz, 8H),2.82 (dd, J=12.5, 2.9 Hz, 2H), 3.09 (dd, J=12.5, 4.9 Hz, 2H), 3.37-3.49(m, 4H), 3.76 (dd, J=4.9, 2.9 Hz, 2H), 5.30-5.40 (m, 4H).

Example 41 (3R,4R)-3,4-bis((Z)-Octadec-11-enyloxy)pyrrolidine (Compound41)

Compound 41 (157 mg, 82.0%) was obtained in the same manner as that inExample 1, by using Compound VI-14 (220 mg, 0.317 mmol) obtained inReference Example 14.

ESI-MS m/z: 605 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.37 (m, 42H), 1.50-1.59 (m, 4H), 1.87 (br s, 2H), 2.01 (q, J=6.1Hz, 8H), 2.83 (dd, J=12.5, 2.9 Hz, 2H), 3.10 (dd, J=12.5, 5.0 Hz, 2H),3.43 (t, J=6.8 Hz, 4H), 3.77 (dd, J=5.0, 2.9 Hz, 2H), 5.30-5.40 (m, 4H).

Example 42 (3R,4R)-3,4-bis((Z)-Icos-11-enyloxy)pyrrolidine (Compound 42)

Compound 42 (168 mg, 84.6%) was obtained in the same manner as that inExample 1, by using Compound VI-15 (225 mg, 0.300 mmol) obtained inReference Example 15.

ESI-MS m/z: 661 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.36 (m, 50H), 1.50-1.59 (m, 4H), 1.78 (br s, 2H), 2.01 (q, J=6.2Hz, 8H), 2.82 (dd, J=12.5, 2.9 Hz, 2H), 3.09 (dd, J=12.5, 5.0 Hz, 2H),3.43 (td, J=6.7, 0.9 Hz, 4H), 3.76 (dd, J=5.0, 2.9 Hz, 2H), 5.30-5.40(m, 4H).

Example 43 (3S,4S)-3,4-bis((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidine(Compound 43)

Compound 43 (728 mg, 90.1%) was obtained in the same manner as that inExample 1, by using Compound VI-10 (929 mg, 1.35 mmol) obtained inReference Example 10.

ESI-MS m/z: 601 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.9 Hz, 6H),1.30-1.41 (m, 30H), 1.50-1.60 (m, 4H), 1.65 (br s, 3H), 2.05 (q, J=6.6Hz, 8H), 2.75-2.85 (m, 6H), 3.09 (dd, J=12.5, 5.3 Hz, 2H), 3.43 (t,J=6.6 Hz, 4H), 3.75-3.78 (m, 2H), 5.28-5.43 (m, 8H).

Example 44 trans-3,4-bis(((Z)-Hexadec-9-enyloxy)methyl)pyrrolidine(Compound 44)

Compound 44 (260 mg, 89.7%) was obtained in the same manner as that inExample 1, by using Compound VI-16 (335 mg, 0.503 mmol) obtained inReference Example 17.

ESI-MS m/z: 577 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.29-1.35 (m, 36H), 1.50-1.59 (m, 4H), 1.96-2.07 (m, 10H), 2.70 (dd,J=11.1, 5.7 Hz, 2H), 3.06 (dd, J=11.1, 7.3 Hz, 2H), 3.28-3.46 (m, 8H),5.30-5.40 (m, 4H).

Example 45 (3R,4R)-3,4-bis(Hexadecyloxy)-1-methylpyrrolidine (Compound45)

Compound 45 (182 mg) was obtained in the same manner as that in Example10, by using Compound 38 (175 mg, 0.317 mmol) obtained in Example 38.

ESI-MS m/z: 567 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.35 (m, 52H), 1.52-1.61 (m, 4H), 2.31 (s, 3H), 2.46 (dd, J=9.6,4.3 Hz, 2H), 2.82 (dd, J=9.6, 5.5 Hz, 2H), 3.37-3.48 (m, 4H), 3.81 (dd,J=5.5, 4.3 Hz, 2H).

Example 46 (3R,4R)-1-Methyl-3,4-bis(octadecyloxy)pyrrolidine (Compound46)

Compound 46 (169 mg, 95.0%) was obtained in the same manner as that inExample 10, by using Compound 39 (174 mg, 0.286 mmol) obtained inExample 39.

ESI-MS m/z: 623 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.35 (m, 60H), 1.52-1.61 (m, 4H), 2.31 (s, 3H), 2.46 (dd, J=9.9,4.3 Hz, 2H), 2.82 (dd, J=9.9, 5.6 Hz, 2H), 3.37-3.48 (m, 4H), 3.81 (dd,J=5.6, 4.3 Hz, 2H).

Example 47 (3R,4R)-1-Methyl-3,4-bis((Z)-tetradec-9-enyloxy)pyrrolidine(Compound 47)

Compound 47 (53.4 mg, 93.6%) was obtained in the same manner as that inExample 10, by using Compound 40 (55 mg, 0.112 mmol) obtained in Example40.

ESI-MS m/z: 507 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.87-0.92 (m, 6H), 1.26-1.35(m, 28H), 1.52-1.61 (m, 4H), 1.98-2.05 (m, 8H), 2.31 (s, 3H), 2.46 (dd,J=9.9, 4.2 Hz, 2H), 2.82 (dd, J=9.9, 5.6 Hz, 2H), 3.37-3.48 (m, 4H),3.81 (dd, J=5.6, 4.2 Hz, 2H), 5.30-5.40 (m, 4H).

Example 48 (3R,4R)-1-Methyl-3,4-bis((Z)-octadec-11-enyloxy)pyrrolidine(Compound 48)

Compound 48 (125 mg, 94.3%) was obtained in the same manner as that inExample 10, by using Compound 41 (130 mg, 0.215 mmol) obtained inExample 41.

ESI-MS m/z: 619 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.36 (m, 44H), 1.52-1.61 (m, 4H), 2.01 (q, J=6.1 Hz, 8H), 2.31 (s,3H), 2.46 (dd, J=9.9, 4.0 Hz, 2H), 2.82 (dd, J=9.9, 5.9 Hz, 2H),3.37-3.48 (m, 4H), 3.81 (t, J=4.9 Hz, 2H), 5.30-5.40 (m, 4H).

Example 49 (3R,4R)-3,4-bis((Z)-Icos-11-enyloxy)-1-methylpyrrolidine(Compound 49)

Compound 49 (132 mg, 91.7%) was obtained in the same manner as that inExample 10, by using Compound 42 (141 mg, 0.214 mmol) obtained inExample 42.

ESI-MS m/z: 675 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.36 (m, 52H), 1.52-1.61 (m, 4H), 2.01 (q, J=6.1 Hz, 8H), 2.31 (s,3H), 2.46 (dd, J=10.0, 4.2 Hz, 2H), 2.82 (dd, J=10.0, 5.9 Hz, 2H),3.37-3.48 (m, 4H), 3.81 (t, J=4.8 Hz, 2H), 5.30-5.40 (m, 4H).

Example 50(3S,4S)-1-Methyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(Compound 50)

Compound 50 (330 mg, 99.2%) was obtained in the same manner as that inExample 10, by using Compound 43 (325 mg, 0.542 mmol) obtained inExample 43.

ESI-MS m/z: 615 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.7 Hz, 6H),1.29-1.38 (m, 30H), 1.51-1.62 (m, 4H), 1.71 (br s, 2H), 2.05 (q, J=6.5Hz, 8H), 2.31 (s, 3H), 2.46 (dd, J=10.0, 4.5 Hz, 2H), 2.75-2.86 (m, 6H),3.36-3.49 (m, 4H), 3.81 (t, J=4.5 Hz, 2H), 5.28-5.43 (m, 8H).

Example 51trans-3,4-bis(((Z)-Hexadec-9-enyloxy)methyl)-1-methylpyrrolidine(Compound 51)

Compound 51 (174 mg, 97.1%) was obtained in the same manner as that inExample 10, by using Compound 44 (175 mg, 0.304 mmol) obtained inExample 44.

ESI-MS m/z: 591 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.26-1.35 (m, 36H), 1.50-1.59 (m, 4H), 1.98-2.11 (m, 10H), 2.33 (s, 3H),2.39 (dd, J=9.2, 5.3 Hz, 2H), 2.67 (dd, J=9.2, 7.3 Hz, 2H), 3.31-3.45(m, 8H), 5.30-5.40 (m, 4H).

Example 52(3S,4S)-1,1-Dimethyl-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidiniumchloride (Compound 52)

Compound 52 (135 mg, 99.8%) was obtained in the same manner as that inExample 19, by using Compound 50 (125 mg, 0.204 mmol) obtained inExample 50.

ESI-MS m/z: 629 M⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.7 Hz, 6H), 1.29-1.38(m, 30H), 1.50-1.59 (m, 4H), 1.72 (br s, 2H), 2.05 (q, J=6.5 Hz, 8H),2.77 (t, J=5.9 Hz, 4H), 3.43-3.57 (m, 4H), 3.68 (s, 6H), 3.87 (dd,J=13.2, 3.6 Hz, 2H), 4.03-4.13 (m, 4H), 5.28-5.44 (m, 8H).

Example 53 (3R,4R)-1-(2,3-diHydroxypropyl)pyrrolidine-3,4-diyldi((9Z,12Z)-octadec-9,12-enoate) (Compound 53)

Compound 53 (95.4 mg, 85.2%) was obtained in the same manner as that inExample 10, by using Compound 2 (100 mg, 0.159 mmol) obtained in Example2 and DL-2,3-dihydroxypropanal (Aldrich; 143 mg, 1.59 mmol).

ESI-MS m/z: 703 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.25-1.40 (m, 28H), 1.56-1.66 (m, 4H), 2.05 (q, J=6.7 Hz, 8H), 2.31 (t,J=7.5 Hz, 4H), 2.39-2.48 (m, 1H), 2.56 (dd, J=10.8, 3.8 Hz, 1H),2.67-2.85 (m, 6H), 3.09 (dd, J=10.3, 5.9 Hz, 1H), 3.24 (dd, J=10.3, 5.9Hz, 1H), 3.50-3.56 (m, 1H), 3.72-3.80 (m, 2H), 5.11 (dd, J=8.8, 5.1 Hz,2H), 5.28-5.43 (m, 8H).

Example 541-((3R,4R)-3,4-bis((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidine-1-yl)propan-2-ol(Compound 54)

Compound 54 (94.7 mg, 86.1%) was obtained in the same manner as that inExample 24, by using Compound 1 (100 mg, 0.167 mmol) obtained in Example1 and 1,2-epoxypropane (0.023 mL, 0.333 mmol),

ESI-MS m/z: 659 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H), 1.12(d, J=6.2 Hz, 3H), 1.26-1.40 (m, 28H), 1.52-1.61 (m, 8H), 2.05 (q, J=6.6Hz, 8H), 2.22-2.30 (m, 1H), 2.41-2.52 (m, 2H), 2.67 (dd, J=10.1, 4.6 Hz,1H), 2.75-2.85 (m, 5H), 3.03 (dd, J=9.5, 5.9 Hz, 1H), 3.38-3.49 (m, 4H),3.73-3.85 (m, 3H), 5.29-5.43 (m, 8H).

Example 551-(3R,4R)-3,4-bis(((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidine-1-yl)-3-methoxypropan-2-ol (Compound 55)

Compound 55 (92.9 mg, 80.8%) was obtained in the same manner as that inExample 24, by using Compound 1 (100 mg, 0.167 mmol) obtained in Example1 and 2-(methoxymethyl)oxirane (0.035 mL, 0.333 mmol)

ESI-MS m/z: 689 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.30-1.38 (m, 28H), 1.52-1.60 (m, 8H), 2.05 (q, J=6.6 Hz, 8H), 2.33-2.43(m, 1H), 2.50-2.56 (m, 1H), 2.59-2.68 (m, 2H), 2.77 (t, J=5.9 Hz, 4H),2.86 (dd, J=9.7, 6.0 Hz, 1H), 3.01 (dd, J=9.7, 6.0 Hz, 1H), 3.33-3.49(m, 9H), 3.79-3.87 (m, 3H), 5.28-5.43 (m, 8H).

Example 563-((3R,4R)-3,4-bis((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidin-1-yl)propan-1-ol(compound 56)

Compound 1 (100 mg, 0.167 mmol) obtained in Example 1 was dissolved inethanol (2 mL), and stirred for 5.5 hours under heat and reflux afteradding ethyl acrylate (0.181 mL, 1.67 mmol) and sodium ethoxide (5.7 mg,0.083 mmol). After cooling the reaction solution, the solvent wasdistilled away under reduced pressure. The resulting residue waspurified by silica gel column chromatography (chloroform/methanol=100/0to 97/3) to give ethyl3-((3R,4R)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)propanoate(107 mg, 91.2%).

The resulting ethyl3-((3R,4R)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)propanoate(220 mg, 0.314 mmol) was dissolved in THF (4 mL), and a 1.0 mol/Ldibutylaluminium hydride toluene solution (0.943 mL, 0.943 mmol) wasadded at −78° C. The mixture was stirred for 2 hours under graduallyincreasing temperatures of −78° C. to 0° C., and a saturated ammoniumchloride aqueous solution was added to the reaction mixture. The aqueouslayer was extracted with ethyl acetate. The organic layer was washedwith saturated brine, dried over magnesium sulfate, and concentratedunder reduced pressure after filtration. The resulting residue waspurified by silica gel column chromatography (chloroform/methanol=100/3to 93/7) to give compound 56 (113 mg, 54.5%).

ESI-MS m/z: 659 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (dd, J=7.5, 6.0 Hz, 6H),1.26-1.40 (m, 32H), 1.50-1.59 (m, 4H), 1.66-1.73 (m, 2H), 2.05 (q, J=6.6Hz, 8H), 2.56-2.79 (m, 8H), 2.90 (dd, J=9.7, 5.7 Hz, 2H), 3.41 (t, J=6.6Hz, 4H), 3.76-3.81 (m, 4H), 5.28-5.43 (m, 8H).

Example 573-((3R,4R)-3,4-bis((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidin-1-yl)propanamide(compound 57)

The ethyl3-((3R,4R)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)propanoate(180 mg, 0.257 mmol) obtained in Example 56 was dissolved in ethanol (2mL). The solution was stirred at room temperature for 2 hours afteradding a 2 mol/L sodium hydroxide aqueous solution (2 mL). The pH wasbrought to 4 by adding a 1 mol/L hydrochloric acid aqueous solution tothe reaction solution, and the aqueous layer was extracted withchloroform. The organic layer was dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure after filtration togive3-((3R,4R)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)propanoicacid (148 mg, 85.8%).

The resulting3-((3R,4R)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)propanoicacid (64 mg, 0.095 mmol) was dissolved in chloroform (2 mL), and stirredovernight at room temperature after addingO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU; Aldrich; 72 mg, 0.190 mmol),diisopropylethylamine (0.083 mL, 0.476 mmol), and a 2 mol/L ammoniamethanol solution (0.238 mL, 0.476 mmol). The aqueous layer wasextracted with ethyl acetate after adding a saturated sodium bicarbonateaqueous solution to the reaction mixture. The organic layer was washedwith water and saturated brine, dried over anhydrous magnesium sulfate,and concentrated under reduced pressure after filtration. The resultingresidue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 90/10) to give compound 57 (46.1 mg,72.1%).

ESI-MS m/z: 672 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.26-1.40 (m, 32H), 1.51-1.60 (m, 4H), 2.05 (q, J=6.6 Hz, 8H), 2.39 (t,J=6.0 Hz, 2H), 2.56 (dd, J=9.7, 4.2 Hz, 2H), 2.63-2.80 (m, 6H), 2.90(dd, J=9.7, 5.9 Hz, 2H), 3.37-3.49 (m, 4H), 3.81 (t, J=4.2 Hz, 2H), 5.24(br s, 1H), 5.28-5.43 (m, 8H), 8.07 (br s, 1H).

Example 583-((3R,4R)-3,4-bis((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidin-1-yl)-N,N-dimethylpropanamide (compound 58)

The3-((3R,4R)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)propanoicacid (63 mg, 0.094 mmol) obtained in Example 57 was dissolved inchloroform (2 mL), and stirred overnight at room temperature afteradding O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU; Aldrich; 71 mg, 0.187 mmol), a 2 mol/Ldimethylamine THF solution (0.234 mL, 0.469 mmol), anddiisopropylethylamine (0.082 mL, 0.469 mmol). The aqueous layer wasextracted with ethyl acetate after adding a saturated sodium bicarbonateaqueous solution to the reaction mixture. The organic layer was washedwith water and saturated brine, dried over anhydrous magnesium sulfate,and concentrated under reduced pressure after filtration. The resultingresidue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 95/5) to give compound 58 (60.7 mg,92.6%).

ESI-MS m/z: 700 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=7.0 Hz, 6H),1.26-1.40 (m, 32H), 1.52-1.61 (m, 4H), 2.05 (q, J=6.6 Hz, 8H), 2.51-2.56(m, 4H), 2.70-2.83 (m, 6H), 2.87-2.95 (m, 2H), 2.93 (s, 3H), 3.00 (s,3H), 3.37-3.49 (m, 4H), 3.81 (t, J=4.8 Hz, 2H), 5.29-5.43 (m, 8H).

Example 59(3R,4R)-1-(3-N,N-Dimethylaminopropyl)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine (compound 59)

Compound 56 (110 mg, 0.167 mmol) obtained in Example 56 was dissolved indichloromethane (3 mL), and stirred at room temperature for 4 hoursafter adding triethylamine (0.082 mL, 0.585 mmol) and anhydrousmethanesulfonic acid (58 mg, 0.334 mmol). The reaction solution waswashed with water and saturated brine, dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure after filtration togive a crude product of3-((3R,4R)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)propylmethanesulfonate.

The resulting crude product was dissolved in THF (1 mL), and irradiatedwith microwave (300 W, 100° C., 1 hour) after adding a 2.0 mol/Ldimethylamine THF solution (0.835 mL, 1.67 mmol). The reaction solutionwas concentrated, and the resulting residue was purified by silica gelcolumn chromatography (chloroform/methanol=100/0 to 75/25) to givecompound 59 (96.6 mg, 84.4%).

ESI-MS m/z: 686 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.26-1.40 (m, 32H), 1.52-1.62 (m, 4H), 1.63-1.76 (m, 2H), 2.05 (q, J=6.7Hz, 8H), 2.23 (s, 6H), 2.29-2.52 (m, 6H), 2.75-2.87 (m, 6H), 3.37-3.49(m, 4H), 3.81 (t, J=4.8 Hz, 2H), 5.28-5.43 (m, 8H).

Example 60

4-((3R,4R)-3,4-bis((9Z,12Z)-Octadec-9,12-dienyloxy)pyrrolidin-1-yl)-1-methylpiperidine (compound 60) Compound 60 (72.0 mg, 62.0%) was obtained in the samemanner as that in Example 10, by using compound 1 (100 mg, 0.167 mmol)obtained in Example 1, and 1-methylpiperidin-4-one (Aldrich; 0.205 mL,1.67 mmol).

ESI-MS m/z: 698 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.29-1.40 (m, 32H), 1.52-1.62 (m, 6H), 1.77-1.83 (m, 2H), 1.89-2.08 (m,11H), 2.24 (s, 3H), 2.53 (dd, J=9.6, 4.7 Hz, 2H), 2.75-2.85 (m, 6H),2.92 (dd, J=9.6, 6.2 Hz, 2H), 3.37-3.49 (m, 4H), 3.81 (t, J=4.7 Hz, 2H),5.28-5.43 (m, 8H).

Example 61(3R,4R)-1-(2-Aminoacetyl)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(compound 61)

Compound 1 (100 mg, 0.167 mmol) obtained in Example 1 was dissolved inchloroform (2 mL), and stirred at room temperature for 4 hours afteradding N-(tert-butoxycarbonyl)glycine (Tokyo Chemical Industry Co.,Ltd.; 55 mg, 0.250 mmol), diisopropylethylamine (0.146 mL, 0.833 mmol),and HATU(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) (Aldrich; 127 mg, 0.333 mmol). The aqueous layerwas extracted with ethyl acetate after adding a saturated sodiumbicarbonate aqueous solution to the reaction mixture. The organic layerwas washed with water and saturated brine, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure afterfiltration. The resulting residue was purified by silica gel columnchromatography (chloroform/methanol=100/0 to 97/3) to give tert-butyl2-((3R,4R)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)-2-oxoethylcarbamate.

The resulting tert-butyl2-((3R,4R)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidin-1-yl)-2-oxoethylcarbamate was dissolved in dichloromethane (2 mL), and stirred at roomtemperature for 1.5 hours after adding trifluoroacetic acid (0.256 mL,3.33 mmol). The aqueous layer was extracted with chloroform after addinga saturated sodium bicarbonate aqueous solution to the reaction mixture.The organic layer was washed with saturated brine, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure afterfiltration. The resulting residue was purified by silica gel columnchromatography (chloroform/methanol=100/0 to 85/15) to give compound 61(60.5 mg, 55.3%).

ESI-MS m/z: 658 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.28-1.40 (m, 32H), 1.49-1.58 (m, 6H), 2.05 (q, J=6.6 Hz, 8H), 2.77 (t,J=5.9 Hz, 4H), 3.30-3.56 (m, 9H), 3.69 (d, J=12.5 Hz, 1H), 3.86 (d,J=4.8 Hz, 1H), 3.92 (d, J=3.7 Hz, 1H), 5.29-5.43 (m, 8H).

Example 62(3R,4R)-1-(3-Aminopropanoyl)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(Compound 62)

Compound 62 (52.5 mg, 49.1%) was obtained in the same manner as that inExample 61, by using Compound 1 (96 mg, 0.160 mmol) obtained in Example1 and N-(tert-butoxycarbonyl)-β-alanine (Tokyo Chemical Industry Co.,Ltd.; 45 mg, 0.240 mmol).

ESI-MS m/z: 672 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=7.0 Hz, 6H),1.26-1.38 (m, 32H), 1.49-1.57 (m, 4H), 1.88 (br s, 2H), 2.05 (q, J=6.7Hz, 8H), 2.43 (td, J=6.0, 1.5 Hz, 2H), 2.77 (t, J=6.0 Hz, 4H), 3.03 (t,J=6.0 Hz, 2H), 3.37-3.67 (m, 8H), 3.85-3.91 (m, 2H), 5.28-5.43 (m, 8H).

Example 63(3R,4R)-1-((S)-2,5-Diaminopentanoyl)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine (Compound 63)

Compound 63 (70.5 mg, 59.4%) was obtained in the same manner as that inExample 61, by using Compound 1 (100 mg, 0.167 mmol) obtained in Example1 and (S)-2,5-bis(tert-butoxycarbonylamino)pentanoic acid (WATANABECHEMICAL INDUSTRIES, LTD.; 83 mg, 0.250 mmol).

ESI-MS m/z: 715 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=7.0 Hz, 6H),1.29-1.68 (m, 40H), 2.05 (q, J=6.7 Hz, 8H), 2.69-2.79 (m, 6H), 3.37-3.56(m, 7H), 3.64-3.69 (m, 2H), 3.85-3.92 (m, 2H), 5.28-5.43 (m, 8H).

Example 64(3R,4R)-1-(6-Aminohexanoyl)-3,4-bis((9Z,12Z)-octadec-9,12-dienyloxy)pyrrolidine(Compound 64)

Compound 64 (85.5 mg, 71.7%) was obtained in the same manner as that inExample 61, by using Compound 1 (100 mg, 0.167 mmol) obtained in Example1 and 6-(tert-butoxycarbonylamino)hexanoic acid (WATANABE CHEMICALINDUSTRIES, LTD.; 58 mg, 0.250 mmol).

ESI-MS m/z: 714 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.26-1.71 (m, 42H), 2.05 (q, J=6.6 Hz, 8H), 2.25 (t, J=7.5 Hz, 2H),2.70-2.79 (m, 6H), 3.37-3.65 (m, 8H), 3.84-3.90 (m, 2H), 5.28-5.43 (m,8H).

Example 65 (3R,4R)-1-(2-aminoacetyl)pyrrolidine-3,4-diyldi((9Z,12Z)-octadec-9,12-enoate) (Compound 65)

Compound 65 (73.4 mg, 67.3%) was obtained in the same manner as that inExample 61, by using Compound 2 (100 mg, 0.159 mmol) obtained in Example2 and N-(tert-butoxycarbonyl)glycine (Tokyo Chemical Industry Co., Ltd.;41.8 mg, 0.239 mmol).

ESI-MS m/z: 686 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.26-1.40 (m, 28H), 1.56-1.65 (m, 4H), 2.05 (q, J=6.7 Hz, 8H), 2.27-2.34(m, 4H), 2.77 (t, J=5.9 Hz, 4H), 3.38-3.53 (m, 3H), 3.68-3.81 (m, 3H),5.20 (br s, 2H), 5.28-5.43 (m, 8H).

Example 66 (3R,4R)-1-((S)-2,6-Diaminohexanoyl)pyrrolidine-3,4-diyldi((9Z,12Z)-octadec-9,12-enoate) (Compound 66)

Compound 66 (73.0 mg, 60.7%) was obtained in the same manner as that inExample 61, by using Compound 2 (100 mg, 0.159 mmol) obtained in Example2 and (S)-2,6-bis(tert-butoxycarbonylamino) hexanoic acid (WATANABECHEMICAL INDUSTRIES, LTD.; 87 mg, 0.239 mmol).

ESI-MS m/z: 757 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.26-1.65 (m, 38H), 2.05 (q, J=6.7 Hz, 8H), 2.28-2.34 (m, 4H), 2.70 (t,J=6.1 Hz, 2H), 2.77 (t, J=6.1 Hz, 4H), 3.43 (dd, J=7.3, 4.8 Hz, 1H),3.54 (d, J=11.9 Hz, 1H), 3.74 (br s, 2H), 3.88 (dd, J=11.9, 4.0 Hz, 1H),5.20-5.21 (m, 2H), 5.29-5.43 (m, 8H).

Example 67 bis(2-((Z)-Octadec-9-enyloxy)ethyl)amine (Compound 67)

Compound 67 (212 mg, 69.5%) was obtained in the same manner as that inExample 1, by using Compound VI-17 (243 mg, 0.349 mmol) obtained inReference Example 19.

ESI-MS m/z: 607 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.25-1.35 (m, 44H), 1.54-1.59 (m, 4H), 1.65 (s, 1H), 1.98-2.05 (m, 8H),2.80 (t, J=5.3 Hz, 4H), 3.42 (t, J=6.8 Hz, 4H), 3.53 (t, J=5.3 Hz, 4H),5.29-5.40 (m, 4H).

Example 68 bis(2-((Z)-Tetradec-9-enyloxy)ethyl)amine (Compound 68)

Compound 68 (291 mg, 85.6%) was obtained in the same manner as that inExample 1, by using Compound VI-18 (402 mg, 0.688 mmol) obtained inReference Example 20.

ESI-MS m/z: 494 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.90 (t, J=7.1 Hz, 6H), 1.29(br s, 28H), 1.53-1.62 (m, 4H), 1.97-2.06 (m, 9H), 2.81 (t, J=5.3 Hz,4H), 3.42 (t, J=6.7 Hz, 4H), 3.53 (t, J=5.4 Hz, 4H), 5.29-5.40 (m, 4H).

Example 69 bis(2-(Tetradecyloxy)ethyl)amine (Compound 69)

Compound 69 (79.2 mg, 62.4%) was obtained in the same manner as that inExample 1, by using Compound VI-19 (150 mg, 0.255 mmol) obtained inReference Example 21.

ESI-MS m/z: 499 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H), 1.26(br s, 44H), 1.52-1.60 (m, 4H), 1.89 (br s, 1H), 2.81 (t, J=5.4 Hz, 4H),3.43 (t, J=6.7 Hz, 4H), 3.54 (t, =5.3 Hz, 4H).

Example 70 bis(2-(Hexadecyloxy)ethyl)amine (Compound 70)

Compound 70 (244 mg, 72.9%) was obtained in the same manner as that inExample 1, by using Compound VI-20 (389 mg, 0.604 mmol) obtained inReference Example 22.

ESI-MS m/z: 555 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H), 1.26(br s, 52H), 1.52-1.61 (m, 4H), 1.91 (br s, 1H), 2.82 (t, J=5.4 Hz, 4H),3.43 (t, J=6.7 Hz, 4H), 3.54 (t, J=5.4 Hz, 4H).

Example 71 bis(2-(Octadecyloxy)ethyl)amine (Compound 71)

Compound 71 (151 mg, 43.4%) was obtained in the same manner as that inExample 1, by using Compound VI-21 (399 mg, 0.570 mmol) obtained inReference Example 23.

ESI-MS m/z: 611 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.7 Hz, 6H), 1.25(br s, 60H), 1.51-1.60 (m, 4H), 2.08 (br s, 1H), 2.84 (t, J=5.3 Hz, 4H),3.43 (t, J=6.6 Hz, 4H), 3.55 (t, J=5.4 Hz, 4H).

Example 72 bis(2-((Z)-Hexadec-9-enyloxy)ethyl)amine (Compound 72)

Compound 72 (516 mg, 85.9%) was obtained in the same manner as that inExample 1, by using Compound VI-22 (700 mg, 1.09 mmol) obtained inReference Example 24.

ESI-MS m/z: 550 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.26-1.35 (m, 36H), 1.52-1.63 (m, 4H), 2.01 (q, J=5.5 Hz, 8H), 2.80 (t,J=5.3 Hz, 4H), 3.42 (t, J=6.6 Hz, 4H), 3.53 (t, J=5.3 Hz, 4H), 5.30-5.40(m, 4H).

Example 73 N,N-bis(2-((Z)-Octadec-9-enoyloxy)ethyl)amine (compound 73)

tert-Butyl bis(2-hydroxyethyl)carbamate (Aldrich; 600 mg, 2.92 mmol) wasdissolved in dichloromethane (30 mL), and stirred overnight at roomtemperature after adding oleic acid (Tokyo Chemical Industry Co., Ltd.;1.82 g, 6.43 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride(1.29 g, 6.72 mmol), and 4-dimethylaminopyridine (89 mg,0.731 mmol). The aqueous layer was extracted with ethyl acetate afteradding a saturated sodium bicarbonate aqueous solution to the reactionmixture. The organic layer was washed with saturated brine, dried overmagnesium sulfate, and concentrated under reduced pressure afterfiltration. The resulting residue was purified by silica gel columnchromatography (hexane/chloroform 50/50 to 0/100) to give tert-butylN,N-bis(2-((Z)-octadec-9-enoyloxy)ethyl)carbamate (1.26 g, 58.7%).

The resulting tert-butylN,N-bis(2-((Z)-octadec-9-enoyloxy)ethyl)carbamate (1.22 g, 1.66 mmol)was dissolved in dichloromethane (30 mL), and stirred at roomtemperature for 3.5 hours after adding trifluoroacetic acid (2.56 mL,33.2 mmol). The aqueous layer was extracted with chloroform after addinga saturated sodium bicarbonate aqueous solution to the reactionsolution. The organic layer was washed with saturated brine, dried overanhydrous magnesium sulfate, and concentrated under reduced pressureafter filtration. The resulting residue was purified by silica gelcolumn chromatography (chloroform/methanol=100/0 to 96/4) to givecompound 73 (998 mg, 94.6%).

ESI-MS m/z: 635 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 40H), 1.58-1.67 (m, 4H), 2.01 (q, J=5.5 Hz, 8H), 2.32 (t,J=7.7 Hz, 4H), 2.89 (t, J=5.5 Hz, 4H), 4.18 (t, J=5.5 Hz, 4H), 5.29-5.40(m, 4H).

Example 74 N,N-bis(2-((9Z,12Z)-Octadec-9,12-dienoyloxy)ethyl)amine(Compound 74)

Compound 74 (494 mg, 82.6%) was obtained in the same manner as that inExample 73, by using tert-butyl bis(2-hydroxyethyl)carbamate (Aldrich;415 mg, 2.02 mmol) and linoleic acid (Aldrich; 1.25 g, 4.45 mmol).

ESI-MS m/z: 631 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.7 Hz, 6H), 1.31(br s, 28H), 1.54 (br s, 1H), 1.58-1.66 (m, 4H), 2.00-2.09 (m, 8H), 2.32(t, J=7.6 Hz, 4H), 2.77 (t, J=5.9 Hz, 4H), 2.89 (t, J=5.4 Hz, 4H), 4.17(t, J=5.6 Hz, 4H), 5.28-5.43 (m, 8H).

Example 75 N-Methyl-N,N-bis(2-((Z)-Tetradec-9-enyloxy)ethyl)amine(Compound 75)

Compound 75 (25.5 mg, 23.9%) was obtained in the same manner as that inReference Example 1, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 25.0 mg, 0.210 mmol) and (Z)-tetradec-9-enylmethanesulfonate(Nu-Chek Prep, Inc; 152 mg, 0.524 mmol).

ESI-MS m/z: 509 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.90 (t, J=7.1 Hz, 6H),1.25-1.37 (m, 28H), 1.51-1.62 (m, 4H), 1.98-2.06 (m, 8H), 2.33 (s, 3H),2.64 (t, J=6.1 Hz, 4H), 3.41 (t, J=6.7 Hz, 4H), 3.52 (t, J=6.1 Hz, 4H),5.29-5.41 (m, 4H).

Example 76 N,N-bis(2-(Hexadecyloxy)ethyl)-N-methylamine (Compound 76)

Compound 76 (135 mg, 47.2%) was obtained in the same manner as that inReference Example 5, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 60.0 mg, 0.504 mmol) and hexadecylmethanesulfonate(Nu-Chek Prep, Inc; 403 mg, 1.26 mmol).

ESI-MS m/z: 569 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H), 1.26(br s, 52H), 1.52-1.61 (m, 4H), 2.33 (s, 3H), 2.64 (t, J=6.0 Hz, 4H),3.41 (t, J=6.8 Hz, 4H), 3.52 (t, J=6.0 Hz, 4H).

Example 77 N-Methyl-N,N-bis(2-((Z)-octadec-11-enyloxy)ethyl)amine(Compound 77)

Compound 77 (198 mg, 47.5%) was obtained in the same manner as that inReference Example 1, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 80 mg, 0.671 mmol) and (Z)-octadec-11-enylmethanesulfonate (Nu-Chek Prep, Inc; 582 mg, 1.68 mmol).

ESI-MS m/z: 621 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.35 (m, 44H), 1.56 (dd, J=16.9, 10.3 Hz, 4H), 2.01 (q, J=5.5 Hz,8H), 2.33 (s, 3H), 2.64 (t, J=6.0 Hz, 4H), 3.41 (t, J=6.8 Hz, 4H), 3.52(t, J=6.0 Hz, 4H), 5.30-5.40 (m, 4H).

Example 78 N,N-bis(2-((Z)-Icos-11-enyloxy)ethyl)-N-methylamine (Compound78)

Compound 78 (164 mg, 45.1%) was obtained in the same manner as that inReference Example 1, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 64.1 mg, 0.538 mmol) and (Z)-icos-11-enylmethanesulfonate (Nu-Chek Prep, Inc; 504 mg, 1.35 mmol).

ESI-MS m/z: 677 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.7 Hz, 6H), 1.27(br s, 52H), 1.50-1.61 (m, 4H), 1.96-2.06 (m, 8H), 2.33 (s, 3H), 2.64(t, J=6.1 Hz, 4H), 3.41 (t, J=6.7 Hz, 4H), 3.52 (t, J=6.1 Hz, 4H),5.29-5.40 (m, 4H).

Example 79N,N-bis(2-((11Z,14Z)-Icos-11,14-dienyloxy)ethyl)-N-methylamine (Compound79)

Compound 79 (204 mg, 62.4%) was obtained in the same manner as that inReference Example 1, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 58.0 mg, 0.487 mmol) and (11Z,14Z)-icos-11,14-dienylmethanesulfonate (Nu-Chek Prep, Inc; 453 mg, 1.22 mmol).

ESI-MS m/z: 673 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.7 Hz, 6H), 1.29(br s, 40H), 1.51-1.60 (m, 4H), 2.01-2.09 (m, 8H), 2.33 (s, 3H), 2.64(t, J=6.1 Hz, 4H), 2.77 (t, J=5.6 Hz, 4H), 3.41 (t, J=6.7 Hz, 4H), 3.52(t, J=6.1 Hz, 4H), 5.28-5.43 (m, 8H).

Example 80 N,N-diMethyl-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)aminiumchloride (Compound 80)

Compound 80 (114 mg, 99.0%) was obtained in the same manner as that inExample 22, by using Compound 67 (104 mg, 0.172 mmol) obtained inExample 67.

ESI-MS m/z: 635 M⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H), 1.27-1.35(m, 44H), 1.51-1.60 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 3.44 (s, 6H), 3.46(t, J=6.6 Hz, 4H), 3.87-3.91 (m, 4H), 3.97-4.01 (m, 4H), 5.29-5.40 (m,4H).

Example 81 N,N-diMethyl-N,N-bis(2-((Z)-octadec-9-enoyloxy)ethyl)aminiumchloride (Compound 81)

Compound 81 (86.9 mg, 79.0%) was obtained in the same manner as that inExample 22, by using Compound 73 (100 mg, 0.158 mmol) obtained inExample 73.

ESI-MS m/z: 663 M⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H), 1.26-1.35(m, 40H), 1.56-1.65 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.35 (t, J=7.7 Hz,4H), 3.53 (s, 6H), 4.12-4.15 (m, 4H), 4.58-4.62 (m, 4H), 5.29-5.40 (m,4H).

Example 82 3-(bis(2-((Z)-Octadec-9-enyloxy)ethyl)amino)propan-1,2-diol(Compound 82)

Compound 82 (24.2 mg, 14.4%) was obtained in the same manner as that inExample 10, by using Compound 67 (150 mg, 0.247 mmol) obtained inExample 67 and DL-2,3-dihydroxypropanal (Aldrich; 223 mg, 2.48 mmol).

ESI-MS m/z: 681 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.36 (m, 44H), 1.52-1.61 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.67-2.69(m, 2H), 2.73-2.88 (m, 4H), 3.41 (t, J=6.8 Hz, 4H), 3.45-3.54 (m, 5H),3.64-3.74 (m, 2H), 5.30-5.40 (m, 4H).

Example 83 3-(bis(2-((Z)-Octadec-9-enoyloxy)ethyl)amino)propan-1,2-diol(Compound 83)

Compound 83 (42.2 mg, 24.2%) was obtained in the same manner as that inExample 10, by using Compound 73 (150 mg, 0.246 mmol) obtained inExample 73 and DL-2,3-dihydroxypropanal (Aldrich; 222 mg, 2.46 mmol).

ESI-MS m/z: 709 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 40H), 1.56-1.66 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.31 (t,J=7.5 Hz, 4H), 2.59-2.72 (m, 2H), 2.76-2.93 (m, 4H), 3.45-3.52 (m, 1H),3.66-3.78 (m, 2H), 4.09-4.21 (m, 4H), 5.29-5.40 (m, 4H).

Example 84 3-(bis(2-((Z)-Octadec-9-enyloxy)ethyl)amino)propanamide(compound 84)

Compound 67 (400 mg, 0.660 mmol) obtained in Example 67 was dissolved inethanol (8 mL), and stirred overnight under heat and reflux after addingethyl acrylate (3.59 mL, 33.0 mmol) and sodium ethoxide (22.5 mg, 0.330mmol). After cooling the reaction solution, the solvent was distilledaway under reduced pressure. The resulting residue was purified bysilica gel column chromatography (chloroform/methanol=100/0 to 98/2) togive ethyl 3-(bis(2-((Z)-octadec-9-enyloxy)ethyl)amino)propanoate (399mg, 85.6%).

The resulting ethyl3-(bis(2-((Z)-octadec-9-enyloxy)ethyl)amino)propanoate (200 mg, 0.283mmol) was dissolved in ethanol (4 mL), and stirred at room temperaturefor 6 hours after adding a 2 mol/L sodium hydroxide aqueous solution (3mL). The pH was brought to 6 by adding a 1 mol/L hydrochloric acidaqueous solution to the reaction solution, and the aqueous layer wasextracted with chloroform. The organic layer was dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure afterfiltration to give 3-(bis(2-((Z)-octadec-9-enyloxy)ethyl)amino)propanoicacid (188 mg, 98.0%).

The resulting 3-(bis(2-((2-octadec-9-enyloxy)ethyl)amino)propanoic acid(85 mg, 0.125 mmol) was dissolved in chloroform (3 mL), and stirredovernight at room temperature after addingO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU; Aldrich; 95 mg, 0.251 mmol), a 7 mol/Lammonia methanol solution (0.090 mL, 0.627 mmol), anddiisopropylethylamine (0.109 mL, 0.627 mmol). The aqueous layer wasextracted with ethyl acetate after adding a saturated sodium bicarbonateaqueous solution to the reaction mixture. The organic layer was washedwith water and saturated brine, dried over anhydrous magnesium sulfate,and concentrated under reduced pressure after filtration. The resultingresidue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 88/12) to give compound 84 (72.3 mg,85.0%).

ESI-MS m/z: 678 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 44H), 1.48-1.57 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.36 (t,J=5.7 Hz, 2H), 2.72-2.80 (m, 6H), 3.39 (t, J=6.8 Hz, 4H), 3.48 (t, J=5.7Hz, 4H), 5.18 (br s, 1H), 5.29-5.40 (m, 4H), 8.22 (br s, 1H).

Example 853-(bis(2-((Z)-Octadec-9-enyloxy)ethyl)amino)-N,N-dimethylpropanamide(compound 85)

The 3-(bis(2-((Z)-octadec-9-enyloxy)ethyl)amino)propanoic acid (80 mg,0.118 mmol) obtained in Example 84 was dissolved in chloroform (3 mL),and stirred overnight at room temperature after addingO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU; Aldrich; 90 mg, 0.236 mmol), a 2 mol/Ldimethylamine THF solution (0.295 mL, 0.590 mmol), anddiisopropylethylamine (0.103 mL, 0.590 mmol). The aqueous layer wasextracted with ethyl acetate after adding a saturated sodium bicarbonateaqueous solution to the reaction mixture. The organic layer was washedwith water and saturated brine, dried over anhydrous magnesium sulfate,and concentrated under reduced pressure after filtration. The resultingresidue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 94/6) to give compound 85 (71.1 mg,85.6%).

ESI-MS m/z: 706 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 44H), 1.51-1.59 (m, 4H), 2.01 (q, J=5.5 Hz, 8H), 2.56 (brs, 2H), 2.79-2.96 (m, 6H), 2.94 (s, 3H), 3.02 (s, 3H), 3.41 (t, J=6.8Hz, 4H), 3.53 (br s, 4H), 5.29-5.40 (m, 4H).

Example 86 2-Amino-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)acetamide(Compound 86)

Compound 86 (83.0 mg, 76.1%) was obtained in the same manner as that inExample 61, by using Compound 67 (100 mg, 0.165 mmol) obtained inExample 67 and N-(tert-butoxycarbonyl)glycine (Tokyo Chemical IndustryCo., Ltd.; 43 mg, 0.247 mmol).

ESI-MS m/z: 664 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 44H), 1.49-1.57 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 3.39 (td,J=6.6, 2.2 Hz, 4H), 3.48-3.51 (m, 4H), 3.53 (s, 2H), 3.56 (s, 4H),5.29-5.40 (m, 4H).

Example 87 3-Amino-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)propanamido(Compound 87)

Compound 87 (84.2 mg, 75.2%) was obtained in the same manner as that inExample 61, by using Compound 67 (100 mg, 0.165 mmol) obtained inExample 67 and N-(tert-butoxycarbonyl)-β-alanine (Tokyo ChemicalIndustry Co., Ltd.; 47 mg, 0.247 mmol).

ESI-MS m/z: 678 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.27-1.35 (m, 44H), 1.49-1.58 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.55 (t,J=6.0 Hz, 2H), 2.99 (t, J=6.0 Hz, 2H), 3.39 (t, J=6.8 Hz, 4H), 3.51-3.57(m, 8H), 5.29-5.40 (m, 4H).

Example 88 6-Amino-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl) (Compound 88)

Compound 88 (87.8 mg, 74.1%) was obtained in the same manner as that inExample 61, by using Compound 67 (100 mg, 0.165 mmol) obtained inExample 67 and 6-(tert-butoxycarbonylamino)hexanoic acid (WATANABECHEMICAL INDUSTRIES, LTD.; 57 mg, 0.247 mmol).

ESI-MS m/z: 720 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.38 (m, 44H), 1.41-1.56 (m, 8H), 1.60-1.70 (m, 2H), 2.01 (q, J=5.5Hz, 8H), 2.39 (t, J=7.5 Hz, 2H), 2.69 (t, J=6.8 Hz, 2H), 3.39 (t, J=6.4Hz, 4H), 3.49-3.56 (m, 8H), 5.29-5.40 (m, 4H).

Example 892-(Dimethylamino)-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)acetamide(Compound 89)

Compound 89 (92.1 mg, 80.8%) was obtained in the same manner as that inExample 25, by using Compound 67 (100 mg, 0.165 mmol) obtained inExample 67 and N,N-dimethylglycine hydrochloride (Tokyo ChemicalIndustry Co., Ltd.; 26 mg, 0.247 mmol).

ESI-MS m/z: 692 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.27-1.35 (m, 44H), 1.49-1.58 (m, 4H), 2.01 (q, J=5.5 Hz, 8H), 2.29 (s,6H), 3.20 (s, 2H), 3.39 (t, J=6.6 Hz, 4H), 3.50-3.57 (m, 6H), 3.68 (t,J=5.5 Hz, 2H), 5.30-5.40 (m, 4H).

Example 903-(Dimethylamino)-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)propanamido(Compound 90)

Compound 90 (53.7 mg, 46.2%) was obtained in the same manner as that inExample 25, by using Compound 67 (100 mg, 0.165 mmol) obtained inExample 67 and 3-(dimethylamino)propionic acid (MATRIX Scientific; 29mg, 0.247 mmol).

ESI-MS m/z: 706 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 44H), 1.49-1.58 (m, 4H), 2.01 (q, J=5.5 Hz, 8H), 2.26 (s,6H), 2.55-2.68 (m, 4H), 3.39 (t, J=6.6 Hz, 4H), 3.50-3.59 (m, 8H),5.30-5.40 (m, 4H).

Example 91(S)-2-Amino-3-hydroxy-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)propanamido(Compound 91)

Compound 91 (31.0 mg, 27.1%) was obtained in the same manner as that inExample 61, by using Compound 67 (100 mg, 0.165 mmol) obtained inExample 67 and (S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanoic acid(WATANABE CHEMICAL INDUSTRIES, LTD.; 51 mg, 0.247 mmol).

ESI-MS m/z: 694 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 44H), 1.49-1.58 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 3.37-3.45(m, 5H), 3.49-3.77 (m, 9H), 3.92 (dd, J=6.4, 4.9 Hz, 1H), 5.29-5.40 (m,4H).

Example 92(S)-2,3-Diamino-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)propanamido(Compound 92)

Compound 92 (91.3 mg, 80.1%) was obtained in the same manner as that inExample 61, by using Compound 67 (100 mg, 0.165 mmol) obtained inExample 67 and (S)-2,3-bis(tert-butoxycarbonylamino)propanoic aciddicyclohexylamine salt (WATANABE CHEMICAL INDUSTRIES, LTD.; 120 mg,0.247 mmol).

ESI-MS m/z: 693 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.28-1.35 (m, 44H), 1.50-1.58 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.70 (dd,J=12.6, 7.3 Hz, 1H), 2.86 (dd, J=12.6, 5.1 Hz, 1H), 3.32-3.43 (m, 5H),3.47-3.58 (m, 5H), 3.71-3.81 (m, 3H), 5.29-5.40 (m, 4H).

Example 93(S)-2,5-Diamino-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)pentanamido(Compound 93)

Compound 93 (57.5 mg, 48.3%) was obtained in the same manner as that inExample 61, by using Compound 67 (100 mg, 0.165 mmol) obtained inExample 67 and (S)-2,5-bis(tert-butoxycarbonylamino)pentanoic acid(WATANABE CHEMICAL INDUSTRIES, LTD.; 82 mg, 0.247 mmol).

ESI-MS m/z: 721 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.27-1.35 (m, 44H), 1.43-1.70 (m, 8H), 2.01 (q, J=5.5 Hz, 8H), 2.68-2.73(m, 2H), 3.29-3.57 (m, 10H), 3.65-3.81 (m, 3H), 5.29-5.40 (m, 4H).

Example 94 (S)-2,6-Diamino-N,N-bis(2-((Z)-octadec-9-enyloxy)ethyl)hexanamido (Compound 94)

Compound 94 (55.7 mg, 46.0%) was obtained in the same manner as that inExample 61, by using Compound 67 (100 mg, 0.165 mmol) obtained inExample 67 and (S)-2,6-bis(tert-butoxycarbonylamino)hexanoic acid(WATANABE CHEMICAL INDUSTRIES, LTD.; 90 mg, 0.247 mmol).

ESI-MS m/z: 735 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.38 (m, 46H), 1.41-1.62 (m, 8H), 2.01 (q, J=5.5 Hz, 8H), 2.69 (t,J=6.6 Hz, 2H), 3.28-3.57 (m, 10H), 3.65-3.82 (m, 3H), 5.29-5.40 (m, 4H).

Example 95 2-Amino-N,N-bis(2-((Z)-octadec-9-enoyloxy)ethyl)acetamide(Compound 95)

Compound 95 (72.8 mg, 66.7%) was obtained in the same manner as that inExample 61, by using Compound 73 (100 mg, 0.158 mmol) obtained inExample 73 and N-(tert-butoxycarbonyl)glycine (Tokyo Chemical IndustryCo., Ltd.; 41.4 mg, 0.237 mmol).

ESI-MS m/z: 692 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 40H), 1.55-1.64 (m, 4H), 2.01 (q, J=5.5 Hz, 8H), 2.30 (td,J=7.7, 2.6 Hz, 4H), 3.51 (s, 2H), 3.53 (t, J=7.0 Hz, 2H), 3.63 (t, J=5.7Hz, 2H), 4.17-4.25 (m, 4H), 5.29-5.40 (m, 4H).

Example 962-Amino-N,N-bis(2-((9Z,12Z)-octadec-9,12-dienoyloxy)ethyl)acetamide(Compound 96)

Compound 96 (36.3 mg, 33.3%) was obtained in the same manner as that inExample 61, by using Compound 74 (100 mg, 0.159 mmol) obtained inExample 74.

ESI-MS m/z: 688 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H), 1.30(br s, 28H), 1.55-1.65 (m, 4H), 2.01-2.08 (m, 8H), 2.27-2.34 (m, 4H),2.77 (t, J=5.9 Hz, 4H), 3.50-3.66 (m, 6H), 4.16-4.27 (m, 4H), 5.28-5.43(m, 8H).

Example 97 (S)-2,6-Diamino-N,N-bis(2-((Z)-octadec-9-enoyloxy)ethyl)hexanamido (Compound 97)

Compound 97 (49.1 mg, 40.8%) was obtained in the same manner as that inExample 61, by using Compound 73 (100 mg, 0.158 mmol) obtained inExample 73 and (S)-2,6-bis(tert-butoxycarbonylamino)hexanoic acid(WATANABE CHEMICAL INDUSTRIES, LTD.; 86 mg, 0.237 mmol).

ESI-MS m/z: 763 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.65 (m, 50H), 2.01 (q, J=5.5 Hz, 8H), 2.27-2.33 (m, 4H), 2.70 (t,J=6.2 Hz, 2H), 3.37-3.56 (m, 2H), 3.64-3.87 (m, 3H), 4.18-4.24 (m, 4H),5.29-5.40 (m, 4H).

Example 982-(Dimethylamino)-N,N-bis(2-((Z)-octadec-9-enoyloxy)ethyl)acetamide(Compound 98)

Compound 98 (72.1 mg, 60.5%) was obtained in the same manner as that inExample 25, by using Compound 73 (105 mg, 0.166 mmol) obtained inExample 73 and N,N-dimethylglycine hydrochloride (Tokyo ChemicalIndustry Co., Ltd.; 25.6 mg, 0.248 mmol).

ESI-MS m/z: 720 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.34 (m, 40H), 1.57-1.64 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.27-2.35(m, 10H), 3.15 (s, 2H), 3.61 (t, J=5.9 Hz, 2H), 3.78 (t, J=5.9 Hz, 2H),4.22 (q, J=5.5 Hz, 4H), 5.30-5.39 (m, 4H).

Example 993-(Dimethylamino)-N,N-bis(2-((Z)-octadec-9-enoyloxy)ethyl)propanamido(Compound 99)

Compound 99 (25.3 mg, 21.9%) was obtained in the same manner as that inExample 25, by using Compound 73 (100 mg, 0.158 mmol) obtained inExample 73 and 3-(dimethylamino)propionic acid (MATRIX Scientific; 36.2mg, 0.309 mmol).

ESI-MS m/z: 734 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 40H), 1.56-1.63 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.27-2.33(m, 10H), 2.52-2.68 (m, 4H), 3.59-3.64 (m, 4H), 4.18-4.23 (m, 4H),5.29-5.40 (m, 4H).

Example 100(S)-2,6-Diamino-N,N-bis(2-((9Z,12Z)-octadec-9,12-dienoyloxy)ethyl)hexanamido (Compound 100)

Compound 100 (85.1 mg, 70.8%) was obtained in the same manner as that inExample 61, by using Compound 74 (100 mg, 0.159 mmol) obtained inExample 74 and (S)-2,6-bis(tert-butoxycarbonylamino)hexanoic acid(WATANABE CHEMICAL INDUSTRIES, LTD.; 87 mg, 0.238 mmol)

ESI-MS m/z: 759 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.26-1.65 (m, 38H), 2.05 (q, J=6.7 Hz, 8H), 2.30 (td, J=7.4, 6.0 Hz,4H), 2.70 (t, J=6.4 Hz, 2H), 2.77 (t, J=5.7 Hz, 4H), 3.37-3.57 (m, 2H),3.64-3.87 (m, 3H), 4.21 (q, J=5.7 Hz, 4H), 5.28-5.44 (m, 8H).

Example 101 trans-3,4-bis(((Z)-Octadec-9-enyloxy)methyl)pyrrolidine(Compound 101)

Compound 101 (252 mg, 80.6%) was obtained in the same manner as that inExample 1, by using Compound VI-23 (357 mg, 0.494 mmol) obtained inReference Example 25.

ESI-MS m/z: 633 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.35 (m, 44H), 1.50-1.59 (m, 4H), 2.01 (q, J=5.5 Hz, 10H), 2.69(dd, J=11.3, 5.5 Hz, 2H), 3.06 (dd, J=11.3, 7.1 Hz, 2H), 3.28-3.46 (m,8H), 5.30-5.40 (m, 4H).

Example 102trans-3,4-bis(((9Z,12Z)-Octadec-9,12-dienyloxy)methyl)pyrrolidine(Compound 102)

Compound 102 (276 mg, 82.7%) was obtained in the same manner as that inExample 1, by using Compound VI-24 (382 mg, 0.532 mmol) obtained inReference Example 26.

ESI-MS m/z: 629 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.29-1.40 (m, 32H), 1.50-1.59 (m, 4H), 1.97-2.08 (m, 10H), 2.69 (dd,J=11.2, 5.7 Hz, 2H), 2.77 (t, J=6.0 Hz, 4H), 3.06 (dd, J=11.2, 7.1 Hz,2H), 3.28-3.46 (m, 8H), 5.29-5.43 (m, 8H).

Example 103trans-3,4-bis(((11Z,14Z)-Icos-11,14-dienyloxy)methyl)pyrrolidine(Compound 103)

Compound 103 (316 mg, 85.2%) was obtained in the same manner as that inExample 1, by using Compound VI-25 (420 mg, 0.542 mmol) obtained inReference Example 27.

ESI-MS m/z: 685 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.27-1.40 (m, 40H), 1.50-1.59 (m, 4H), 1.97-2.08 (m, 10H), 2.69 (dd,J=11.1, 5.8 Hz, 2H), 2.77 (t, J=5.8 Hz, 4H), 3.06 (dd, J=11.1, 7.3 Hz,2H), 3.28-3.46 (m, 8H), 5.28-5.43 (m, 8H).

Example 104 trans-3,4-bis(((E)-Octadec-9-enoyloxy)methyl)pyrrolidine(compound 104)

Compound XIII-1 (278 mg, 0.366 mmol) obtained in Reference Example 28was dissolved in dichloromethane (6 mL), and stirred at room temperaturefor 3 hours after adding trifluoroacetic acid (0.563 mL, 7.31 mmol). Theaqueous layer was extracted with chloroform after adding a saturatedsodium bicarbonate aqueous solution to the reaction mixture. The organiclayer was washed with saturated brine, dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure after filtration. Theresulting residue was dissolved in a small amount of methanol, andadsorbed on the upper part of BONDESIL-SCX (VARIAN; 6 g) charged into aplastic column. After washing with methanol, the target was eluted withan ammonia.methanol solution (Tokyo Chemical Industry Co., Ltd.; 2mol/L). The fraction comprising the target was concentrated underreduced pressure to give compound 104 (162 mg, 67.2%).

ESI-MS m/z: 661 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.27-1.35 (m, 40H), 1.56-1.64 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.09-2.16(m, 2H), 2.30 (t, J=7.5 Hz, 4H), 2.72 (dd, J=11.3, 5.5 Hz, 2H), 3.11(dd, J=11.3, 7.1 Hz, 2H), 3.99-4.12 (m, 4H), 5.29-5.40 (m, 4H).

Example 105trans-3,4-bis(((9Z,12Z)-Octadec-9,12-dienoyloxy)methyl)pyrrolidine(Compound 105)

Compound 105 (224 mg, 73.6%) was obtained in the same manner as that inExample 104, by using Compound XIII-2 (350 mg, 0.463 mmol) obtained inReference Example 29.

ESI-MS m/z: 657 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.26-1.40 (m, 28H), 1.57-1.66 (m, 4H), 2.05 (q, J=6.6 Hz, 8H), 2.09-2.17(m, 2H), 2.31 (t, J=7.5 Hz, 4H), 2.72 (dd, J=11.3, 6.0 Hz, 2H), 2.77 (t,J=6.2 Hz, 4H), 3.11 (dd, J=11.3, 7.3 Hz, 2H), 3.99-4.13 (m, 4H),5.28-5.43 (m, 8H).

Example 106trans-1-Methyl-3,4-bis(((Z)-octadec-9-enyloxy)methyl)pyrrolidine(Compound 106)

Compound 106 (87.3 mg, 79.9%) was obtained in the same manner as that inExample 10, by using Compound 101 (107 mg, 0.169 mmol) obtained inExample 101.

ESI-MS m/z: 647 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H),1.27-1.36 (m, 44H), 1.50-1.59 (m, 4H), 1.98-2.09 (m, 10H), 2.31 (s, 3H),2.36 (dd, J=9.2, 5.3 Hz, 2H), 2.64 (dd, J=9.2, 7.0 Hz, 2H), 3.30-3.45(m, 8H), 5.29-5.40 (m, 4H).

Example 107trans-1-Methyl-3,4-bis(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)pyrrolidine(Compound 107)

Compound 107 (109 mg, 86.7%) was obtained in the same manner as that inExample 10, by using Compound 102 (123 mg, 0.196 mmol) obtained inExample 102.

ESI-MS m/z: 643 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.26-1.40 (m, 32H), 1.50-1.60 (m, 4H), 2.05 (q, J=6.6 Hz, 10H), 2.31 (s,3H), 2.36 (dd, J=9.2, 5.5 Hz, 2H), 2.64 (dd, J=9.2, 7.0 Hz, 2H), 2.77(t, J=5.9 Hz, 4H), 3.30-3.45 (m, 8H), 5.28-5.43 (m, 8H).

Example 108trans-3,4-bis(((11Z,14Z)-Icos-11,14-dienyloxy)methyl)-1-methylpyrrolidine(Compound 108)

Compound 108 (145 mg, 85.9%) was obtained in the same manner as that inExample 10, by using Compound 103 (165 mg, 0.241 mmol) obtained inExample 103.

ESI-MS m/z: 699 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.27-1.40 (m, 40H), 1.50-1.60 (m, 4H), 2.05 (q, J=6.2 Hz, 10H), 2.31 (s,3H), 2.36 (dd, J=9.2, 5.8 Hz, 2H), 2.64 (dd, J=9.2, 7.3 Hz, 2H), 2.77(t, J=5.8 Hz, 4H), 3.31-3.45 (m, 8H), 5.29-5.43 (m, 8H).

Example 109trans-1-Methyl-3,4-bis(((Z)-octadec-9-enoyloxy)methyl)pyrrolidine(Compound 109)

Compound 109 (47 mg, 92%) was obtained in the same manner as that inExample 10, by using Compound 104 (50 mg, 0.076 mmol) obtained inExample 104.

ESI-MS m/z: 675 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.26-1.35 (m, 40H), 1.56-1.65 (m, 4H), 2.01 (q, J=5.5 Hz, 8H), 2.15-2.24(m, 2H), 2.27-2.37 (m, 9H), 2.67 (dd, J=9.3, 7.1 Hz, 2H), 3.99-4.12 (m,4H), 5.29-5.40 (m, 4H).

Example 110trans-1-Methyl-3,4-bis(((9Z,12Z)-octadec-9,12-dienoyloxy)methyl)pyrrolidine(Compound 110)

Compound 110 (66 mg, 81%) was obtained in the same manner as that inExample 10, by using Compound 105 (80 mg, 0.12 mmol) obtained in Example105.

ESI-MS m/z: 671 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.25-1.40 (m, 28H), 1.57-1.66 (m, 4H), 2.05 (q, J=6.7 Hz, 8H), 2.13-2.24(m, 2H), 2.27-2.37 (m, 9H), 2.66 (dd, J=9.2, 7.3 Hz, 2H), 2.77 (t, J=5.7Hz, 4H), 3.99-4.12 (m, 4H), 5.28-5.43 (m, 8H).

Example 111trans-1,1-Dimethyl-3,4-bis(((Z)-octadec-9-enyloxy)methyl)pyrrolidiniumchloride (Compound 111)

Compound 111 (85.9 mg, 86.6%) was obtained in the same manner as that inExample 22, by using Compound 101 (90.0 mg, 0.142 mmol) obtained inExample 101.

ESI-MS m/z: 661 M⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, =6.6 Hz, 6H), 1.27-1.37(m, 44H), 1.50-1.58 (m, 4H), 2.01 (q, J=5.9 Hz, 8H), 2.82-2.87 (m, 2H),3.43 (t, J=6.6 Hz, 4H), 3.48 (s, 6H), 3.49-3.56 (m, 4H), 3.75 (dd,J=11.6, 8.2 Hz, 2H), 4.10 (dd, J=11.6, 8.1 Hz, 2H), 5.29-5.41 (m, 4H).

Example 112trans-1,1-Dimethyl-3,4-bis(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)pyrrolidiniumchloride (Compound 112)

Compound 112 (107 mg, 96.9%) was obtained in the same manner as that inExample 22, by using Compound 102 (100 mg, 0.159 mmol) obtained inExample 102.

ESI-MS m/z: 657 M⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H), 1.29-1.40(m, 32H), 1.50-1.58 (m, 4H), 2.05 (q, J=6.7 Hz, 8H), 2.77 (t, J=5.9 Hz,4H), 2.80-2.87 (m, 2H), 3.43 (t, J=6.6 Hz, 4H), 3.48 (s, 6H), 3.49-3.56(m, 4H), 3.74 (dd, J=11.6, 8.1 Hz, 2H), 4.09 (dd, J=11.6, 8.1 Hz, 2H),5.28-5.43 (m, 8H).

Example 113trans-1,1-Dimethyl-3,4-bis(((Z)-octadec-9-enoyloxy)methyl)pyrrolidiniumchloride (Compound 113)

Compound 113 (69.5 mg, 82.0%) was obtained in the same manner as that inExample 22, by using Compound 104 (77.0 mg, 0.117 mmol) obtained inExample 104.

ESI-MS m/z: 689 M⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H), 1.27-1.35(m, 40H), 1.56-1.65 (m, 4H), 2.01 (q, J=5.5 Hz, 8H), 2.33 (t, J=7.5 Hz,4H), 2.93 (br s, 2H), 3.57 (s, 6H), 3.86 (dd, J=11.9, 8.6 Hz, 2H),4.19-4.27 (m, 6H), 5.29-5.40 (m, 4H).

Example 114trans-1,1-Dimethyl-3,4-bis(((9Z,12Z)-octadec-9,12-dienoyloxy)methyl)pyrrolidiniumchloride (Compound 114)

Compound 114 (74.3 mg, 64.4%) was obtained in the same manner as that inExample 22, by using Compound 105 (105 mg, 0.160 mmol) obtained inExample 105.

ESI-MS m/z: 685 M⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=7.0 Hz, 6H), 1.26-1.40(m, 28H), 1.57-1.65 (m, 4H), 2.05 (q, J=6.7 Hz, 8H), 2.33 (t, J=7.7 Hz,4H), 2.77 (t, J=5.9 Hz, 4H), 2.93 (br s, 2H), 3.56 (s, 6H), 3.86 (dd,J=12.1, 8.4 Hz, 2H), 4.19-4.27 (m, 6H), 5.28-5.43 (m, 8H).

Example 115trans-1-((S)-2,6-Diaminohexanoyl)-3,4-bis(((11Z,14Z)-icos-11,14-dienyloxy)methyl)pyrrolidine (Compound 115)

Compound 115 (72.6 mg, 61.2%) was obtained in the same manner as that inExample 61, by using Compound 103 (100 mg, 0.146 mmol) obtained inExample 103 and (S)-2,6-bis(tert-butoxycarbonylamino)hexanoic acid(WATANABE CHEMICAL INDUSTRIES, LTD.; 80 mg, 0.219 mmol).

ESI-MS m/z: 813 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=7.0 Hz, 6H),1.27-1.59 (m, 50H), 2.05 (q, J=6.6 Hz, 8H), 2.17-2.26 (m, 1H), 2.35-2.43(m, 1H), 2.70 (t, J=5.7 Hz, 2H), 2.77 (t, =6.0 Hz, 4H), 3.21-3.55 (m,11H), 3.58-3.80 (m, 2H), 5.29-5.43 (m, 8H).

Example 116 3-(Dimethylamino)propylbis(2-((Z)-1-oxooctadec-9-enyloxy)ethyl)carbamate (compound 116)

Compound 73 (160 mg, 0.252 mmol) obtained in Example 73 was dissolved inchloroform (2.5 mL), and heat-stirred at 110° C. for 30 minutes with amicrowave reactor after adding 3-(dimethylamino)propyl 4-nitrophenylcarbonate hydrochloride (115 mg, 0.379 mmol) synthesized according tothe method described in Journal of American Chemical Society (J. Am.Chem. Soc.), 1981, Vol. 103, p. 4194-4199, and triethylamine (0.141 mL,1.01 mmol). 3-(Dimethylamino)propyl 4-nitrophenyl carbonatehydrochloride (38.4 mg, 0.126 mmol) was added to the reaction mixture,and heat-stirred at 110° C. for 30 minutes with a microwave reactor.After being diluted with chloroform, the reaction mixture was washedwith a 1 mol/L sodium hydroxide aqueous solution three times and thenwith saturated brine, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure after filtration. The resultingresidue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 95/5) to give compound 116 (42.6 mg,22.1%).

ESI-MS m/z: 764 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.24-1.37 (m, 40H), 1.54-1.65 (m, 4H), 1.78-1.89 (m, 2H), 1.97-2.05 (m,8H), 2.23 (s, 6H), 2.25-2.38 (m, 6H), 3.48-3.58 (m, 2H), 3.62 (q, J=5.2Hz, 2H), 4.11-4.30 (m, 6H), 5.28-5.41 (m, 4H).

Example 117 3-(Dimethylamino)propylbis(2-((9Z,12Z)-1-oxooctadec-9,12-dienyloxy)ethyl)carbamate (compound117)

Compound 117 (60.3 mg, 31.5%) was obtained in the same manner as that inExample 116, by using compound 74 (159 mg, 0.252 mmol) obtained inExample 74.

ESI-MS m/z: 760 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.9 Hz, 6H),1.26-1.38 (m, 28H), 1.58-1.69 (m, 4H), 1.78-1.89 (m, 2H), 2.00-2.09 (m,8H), 2.23 (s, 6H), 2.26-2.38 (m, 6H), 2.77 (t, J=5.8 Hz, 4H), 3.48-3.57(m, 2H), 3.62 (q, J=5.3 Hz, 2H), 4.12-4.30 (m, 6H), 5.28-5.44 (m, 8H).

Compounds 118 to 136 can be obtained by using the same methods used inExamples 1 to 117, or by using the method described in WO2009/086558.

Reference Example 30 (3R,4R)-1-Methylpyrrolidine-3,4-diyldi((9Z,12Z)-octadec-9,12-dienoate) (Compound A-3)

Compound A-3 (1.17 g, 95.4%) was obtained in the same manner as that inExample 10, by using Compound 2 (1.20 g, 1.90 mmol) obtained in Example2.

ESI-MS m/z: 643 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=7.0 Hz, 6H),1.31-1.41 (m, 28H), 1.56-1.66 (m, 4H), 2.05 (q, =6.6 Hz, 8H), 2.29-2.35(m, 7H), 2.48 (dd, J=10.3, 4.2 Hz, 2H), 2.77 (t, J=5.8 Hz, 4H), 3.04(dd, J=10.3, 5.8 Hz, 2H), 5.11 (dd, J=5.8, 4.2 Hz, 2H), 5.28-5.43 (m,8H).

Reference Example 31 (3R,4R)-1-Methylpyrrolidine-3,4-diyldi((Z)-octadec-9-enoate) (Compound A-4)

Compound A-4 (481 mg, 94.0%) was obtained in the same manner as that inExample 10, by using Compound 9 (500 mg, 0.791 mmol) obtained in Example9.

ESI-MS m/z: 647 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.88 (t, J=6.6 Hz, 6H),1.27-1.35 (m, 40H), 1.56-1.66 (m, 4H), 2.01 (q, J=6.2 Hz, 8H), 2.32 (t,J=7.7 Hz, 4H), 2.35 (s, 3H), 2.48 (dd, J=10.5, 4.0 Hz, 2H), 3.04 (dd,J=10.5, 5.7 Hz, 2H), 5.10 (dd, J=5.7, 4.0 Hz, 2H), 5.29-5.40 (m, 4H).

Reference Example 32N-Methyl-N,N-bis(2-((9Z,12Z)-1-oxooctadec-9,12-dienyloxy)ethyl)amine(Compound A-5)

Compound A-5 (348 mg, 54.0%) was obtained in the same manner as that inReference Example 2, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 119 mg, 1.00 mmol) and linoleic acid(Aldrich; 617mg, 2.20 mmol).

ESI-MS m/z: 645 (M+H)⁺; ¹H-NMR (CDCl₃) δ: 0.89 (t, J=6.8 Hz, 6H),1.27-1.38 (m, 28H), 1.56-1.66 (m, 4H), 2.00-2.09 (m, 8H), 2.31 (t, =7.6Hz, 4H), 2.35 (s, 3H), 2.70 (t, J=5.9 Hz, 4H), 2.77 (t, J=5.8 Hz, 4H),4.16 (t, J=5.9 Hz, 4H), 5.28-5.42 (m, 8H).

Reference Example 33N-Methyl-N,N-bis(2-((Z)-1-oxooctadec-9-enyloxy)ethyl)amine (CompoundA-6)

Compound A-6 (333 mg, 51.4%) was obtained in the same manner as that inReference Example 2, by using N-methyldiethanolamine (Tokyo ChemicalIndustry Co., Ltd.; 119 mg, 1.00 mmol) and oleic acid (Tokyo ChemicalIndustry Co., Ltd.; 621 mg, 2.20 mmol) ESI-MS m/z: 649 (M+H)⁺; ¹H-NMR(CDCl₃) δ: 0.88 (t, J=6.8 Hz, 6H), 1.25-1.36 (m, 40H), 1.56-1.67 (m,4H), 1.97-2.04 (m, 8H), 2.30 (t, =7.6 Hz, 4H), 2.35 (s, 3H), 2.70 (t,J=5.9 Hz, 4H), 4.16 (t, J=5.9 Hz, 4H), 5.28-5.39 (m, 4H).

The composition of the present invention is described below in detailusing Examples and Test Examples. It should be noted that the presentinvention is in no way limited by the following Examples and TestExamples.

Example 118

Preparations were produced using the compounds obtained in Examples 1 to117, as follows.

Anti-APO-B siRNA was used as the nucleic acid. Anti-APO-B siRNAsuppresses expression of an apolipoprotein-B (hereinafter, “apo-b”) geneand has a sense strand with the base sequence5′-GmUCAmUCACACmUGAAmUACCAAmU-3′ (the sugars attached to the basesappended with m are 2′-O-methyl-substituted riboses), and an antisensestrand with the base sequence 5′-AUUGGUAUUCAGUGUGAUGACAC-3′ (the 5′-endis phosphorylated). The sense strand and the antisense strand wereobtained from Nippon EGT or Hokkaido System Science Co., Ltd., andannealed to prepare the nucleic acid (hereinafter, “apo-b siRNA”).

A solution comprising the constituent components of a lipid membrane wasprepared by dissolving each of the weighed samples in 90 vol % ethanolin 8.947/1.059/5.708/13.697 mmol/L [compounds 1 to 117 obtained inExamples 1 to117/1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethyleneglycol)-2000) (PEG-DMPE, N-(carbonylmethoxypolyethylene glycol2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine sodium salt, NOFCorporation)/distearoylphosphatidyl choline (DSPC,1,2-distearoyl-sn-glycero-3-phosphocholine, NOF Corporation)/cholesterol(Avanti Polar Lipids)]. Separately, apo-b siRNA/distilled water (24mg/mL) was diluted with a Tris-EDTA buffer (200 mM Tris-HCl, 20 mM EDTA,Invitrogen) and a 20 mM citric acid buffer (pH 5.0) to prepare a 1.5mg/mL apo-b siRNA aqueous solution (2 mM Tris-EDTA buffer, pH 5.0).

The resulting lipid solution was heated to 37° C., and a 100-μL portionwas transferred to a preparation container. The apo-b siRNA aqueoussolution (100 μL) was then added thereto while being stirred. Then, a 20mM citric acid buffer (containing 300 mM NaCl, pH 6.0; 200 μL) was addedto the lipid nucleic acid mixed suspension (200 μL) while being stirred.The siRNA concentration was brought to 10 μM by dropping a Dulbeccophosphate buffer (DPBS, Invitrogen; 662 μL), and preparations(compositions comprising compounds 1 to 117 and the nucleic acid) wereobtained.

The average particle diameter of the liposomes in each preparation wasmeasured with a particle diameter measurement device (Malvern; ZetasizerNano ZS). The results are presented in Table 18.

TABLE 18 Compound No. 1 2 3 4 5 6 7 8 9 10 Particle diameter of 137.4139.6 152.8 160.6 154.2 140.3 148.7 143.8 141.9 153.9 Preparetion (nm)Compound No. 11 12 13 14 15 16 17 18 19 20 Particle diameter of 135.8161.3 150.2 139.3 139.1 143.2 142.6 146.4 122.4 107.5 Preparetion (nm)Compound No. 21 22 23 24 25 26 27 28 29 30 Particle diameter of 112.4115.6 108.7 160.7 126.0 133.9 135.2 132.2 122.5 132.7 Preparetion (nm)Compound No. 31 32 33 34 35 36 37 38 39 40 Particle diameter of 100.7154.2 152.1 165.7 126.5 137.2 151.3 157.2 444.5 133.8 Preparetion (nm)Compound No. 41 42 43 44 45 46 47 48 49 50 Particle diameter of 134.4131.3 150.0 143.5 136.3 192.0 125.8 135.0 141.7 129.2 Preparetion (nm)Compound No. 51 52 53 54 55 56 57 58 59 60 Particle diameter of 123.9118.9 152.4 148.9 139.6 120.3 159.2 151.8 116.7 125.3 Preparetion (nm)Compound No. 61 62 63 64 65 66 67 68 69 70 Particle diameter of 125.9151.3 143.0 146.0 128.9 137.4 127.6 132.4 136.8 145.6 Preparetion (nm)Compound No. 71 72 73 74 75 76 77 78 79 80 Particle diameter of 148.4130.6 146.8 128.4 124.0 133.6 119.7 131.4 116.5 110.3 Preparetion (nm)Compound No. 81 82 83 84 85 86 87 88 89 90 Particle diameter of 103.4125.7 169.1 120.2 120.1 128.4 136.3 131.9 121.5 120.8 Preparetion (nm)Compound No. 91 92 93 94 95 96 97 98 99 100 Particle diameter of 125.4143.9 135.1 137.3 128.3 133.2 133.0 124.1 129.6 135.1 Preparetion (nm)Compound No. 101 102 103 104 105 106 107 108 109 110 Particle diameterof 148.7 133.0 141.8 141.5 125.6 139.2 139.1 144.9 129.6 131.6Preparetion (nm) Compound No. 111 112 113 114 115 116 117 Particlediameter of 112.0 119.2 103.5 108.2 152.8 154.1 155.9 Preparetion (nm)

Comparative Example 1

A preparation was obtained in the same manner as that in Example 118,except that compound 1 was changed to DOTAP (compound A-1, Avanti PolarLipids). The average particle diameter of the liposomes in thepreparation was 104.0 nm.

Comparative Example 2

A preparation was obtained in the same manner as that in Example 118,except that compound 1 was changed to DLinDMA (Compound A-2). TheCompound A-2 was produced by a method described in WO2005/121348. Theaverage particle diameter of the liposomes in the preparation was 131.6nm.

Comparative Example 3

A preparation was obtained in the same manner as that in Example 118 byusing Compound A-3 obtained in Reference Example 30. The averageparticle diameter of the liposomes in the preparation was 141.0 nm.

Comparative Example 4

A preparation was obtained in the same manner as that in Example 118 byusing Compound A-4 obtained in Reference Example 31. The averageparticle diameter of the liposomes in the preparation was 131.1 nm.

Comparative Example 5

A preparation was obtained in the same manner as that in Example 118 byusing Compound A-5 obtained in Reference Example 32. The averageparticle diameter of the liposomes in the preparation was 136.4 nm.

Comparative Example 6

A preparation was obtained in the same manner as that in Example 118 byusing Compound A-6 obtained in Reference Example 33. The averageparticle diameter of the liposomes in the preparation was 139.5 nm.

Comparative Example 7

A preparation was obtained in the same manner as that in Example 118 byusing Compound VI-3 obtained in Reference Example 3. The averageparticle diameter of the liposomes in the preparation was 167.8 nm.

Comparative Example 8

A preparation was obtained in the same manner as that in Example 118 byusing Compound VI-4 obtained in Reference Example 4. The averageparticle diameter of the liposomes in the preparation was 157.8 nm.

The structures of Compound A-1 to 6 and Compound VI-3 to 4 used inComparative Examples are shown in Tables 19.

TABLE 19 Compound No. Structure A-1

A-2

A-3

A-4

A-5

A-6

VI-3

VI-4

Test Example 1

The preparations obtained in Example 118 (compositions comprisingcompounds 1 to 115 and the nucleic acid), and the preparations obtainedin Comparative Examples 1 to 8 were introduced into human livercancer-derived cell line HepG2 (HB-8065) by using the following method.

Each preparation diluted with Opti-MEM (GIBCO; 31985) to make thenucleic acid final concentrations 3 to 100 nM was dispensed in a 96-wellculture plate in 20-μL portions. Then, HepG2 cells suspended in MEMcontaining 1.25% fetal bovine serum (FBS; SAFC Biosciences; 12203C) wereinoculated in 6250 cells/80 μL/well, and cultured under 37° C., 5% CO₂conditions to introduce the preparation into the HepG2 cells. Untreatedcells were also inoculated as a negative control group.

The cells after the introduction of the preparation were cultured in a37° C., 5% CO₂ incubator for 24 hours, and washed with ice-cooledphosphate buffered saline (PBS; GIBCO; 14190). Total RNA was collected,and cDNA was produced by reverse transcription reaction using the totalRNA as a template, using a Cells-to-Ct Kit (Applied Bioscience; ABI;AM1728) according to the protocol attached to the kit.

By using the cDNA as a template, a PCR reaction was performed for theapo-b gene and the constitutively expressed geneD-glyceraldehyde-3-phosphate dehydrogenase (hereinafter, “gapdh”) geneusing a universal probe library (Roche Applied Science; 04683633001) asthe probe. For the PCR, ABI7900HT Fast (ABI) was used according to theprotocol attached to the system. The mRNA amplification amounts weremeasured, and a quasi-quantitative value for the apo-b mRNA wascalculated using the gapdh mRNA amplification amount as the internalcontrol. The apo-b mRNA level and the gapdh mRNA amplification amount inthe negative control group were also measured in the same manner, and aquasi-quantitative value for the apo-b mRNA was calculated using thegapdh mRNA amplification amount as the internal control.

The apo-b mRNA expression rate was determined from the calculated apo-bmRNA quasi-quantitative value relative to the apo-b mRNAquasi-quantitative value of the negative control as 1. The results forExample 118 are presented in FIG. 1 to 12, and the results forComparative Examples 1 to 8 are presented in FIG. 13. The vertical axisrepresents the target gene mRNA expression rate relative to the negativecontrol taken at 1. The horizontal axis represents nucleic acidconcentration (nM), and the compound numbers and example numbers of thecationic lipids used.

As is clear FIG. 1 to 12, the apo-b gene mRNA expression rate wassuppressed after the introduction of the preparations obtained inExample 118 (compositions comprising the apo-b geneexpression-suppressing anti-APO-B siRNA, and compounds 1 to 115) intothe human liver cancer-derived cell line HepG2. On the other hand, as isclear FIG. 13, the apo-b gene mRNA expression rate was not suppressedafter the introduction of the preparations obtained in ComparativeExamples 3 to 8 (compositions comprising the apo-b geneexpression-suppressing anti-APO-B siRNA, and compounds A-3 to 6, VI-3and VI-4) into the human liver cancer-derived cell line HepG2.

It was therefore found that the composition of the present invention canbe used to introduce nucleic acid into cells and the like, and that thecationic lipid of the present invention represents a novel cationiclipid that allows nucleic acid to be easily introduced into cells.

INDUSTRIAL APPLICABILITY

A composition comprising the novel cationic lipid of the presentinvention and a nucleic acid can be used to easily introduce the nucleicacid into, for example, cells and the like through administration tomammals and the like.

SEQUENCE LISTING FREE TEXT

SEQ No. 1: siRNA senseSEQ No. 2: siRNA antisenseSEQ No. 2: 5′-phosphorylated Adenosine

SEQUENCE LISTING

Apo-b siNA.txt

1-27. (canceled)
 28. A cationic lipid represented by formula (I):

wherein: R¹ and R² are, the same or different, each linear or branchedalkyl, alkenyl or alkynyl having 12 to 24 carbon atoms, or R¹ and R² arecombined together to form dialkylmethylene, dialkenylmethylene,dialkynylmethylene or alkylalkenylmethylene, X¹ and X² are hydrogenatoms, X³ is absent or is alkyl having 1 to 6 carbon atoms, or alkenylhaving 3 to 6 carbon atoms, when X³ is absent, Y is absent, a and b are0, L³ is a single bond, R³ is alkyl having 1 to 6 carbon atoms, alkenylhaving 3 to 6 carbon atoms, pyrrolidin-3-yl, piperidin-3-yl,piperidin-4-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are),the same or different, amino, monoalkylamino, dialkylamino,trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl,dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, and L¹ and L²are —O—, Y is absent, a and b are, the same or different, 0 to 3, andare not 0 at the same time, L³ is a single bond, R³ is alkyl having 1 to6 carbon atoms, alkenyl having 3 to 6 carbon atoms, pyrrolidin-3-yl,piperidin-3-yl, piperidin-4-yl, or alkyl having 1 to 6 carbon atoms oralkenyl having 3 to 6 carbon atoms substituted with 1 to 3substituent(s), which is(are), the same or different, amino,monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidylor morpholinyl, L¹ and L² are, the same or different, —O—, —CO—O— or—O—CO—, or Y is absent, a and b are, the same or different, 0 to 3, L³is —CO— or —CO—O—, R³ is pyrrolidin-2-yl, pyrrolidin-3-yl,piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl,morpholin-3-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are),the same or different, amino, monoalkylamino, dialkylamino,trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl,dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, wherein atleast one of the substituents is amino, monoalkylamino, dialkylamino,trialkylammonio, pyrrolidinyl, piperidyl or morpholinyl, and L¹ and L²are, the same or different, —O—, —CO—O— or —O—CO—, and when X³ is alkylhaving 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms, Y is apharmaceutically acceptable anion, a and b are, the same or different, 0to 3, L³ is a single bond, R³ is alkyl having 1 to 6 carbon atoms,alkenyl having 3 to 6 carbon atoms, pyrrolidin-3-yl, pyrrolidin-2-yl,pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl,morpholin-2-yl, morpholin-3-yl, or alkyl having 1 to 6 carbon atoms oralkenyl having 3 to 6 carbon atoms substituted with 1 to 3substituent(s), which is(are), the same or different, amino,monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidylor morpholinyl, L¹ and L² are, the same or different, —O—, —CO—O— or—O—CO—.
 29. The cationic lipid according to claim 28, wherein L¹ and L²are —O— or —O—CO—, and R¹ and R² are dodecyl, tetradecyl, hexadecyl,octadecyl, icosyl, docosyl, tetracosyl, (Z)-tetradec-9-enyl,(Z)-hexadec-9-enyl, (Z)-octadec-6-enyl, (Z)-octadec-9-enyl,(E)-octadec-9-enyl, (Z)-octadec-11-enyl, (9Z,12Z)-octadec-9,12-dienyl,(9Z,12Z,15Z)-octadec-9,12,15-trienyl, (Z)-icos-11-enyl,(11Z,14Z)-icos-11,14-dienyl, 3,7,11-trimethyldodeca-2,6,10-trienyl or3,7,11,15-tetramethylhexadec-2-enyl.
 30. The cationic lipid according toclaim 28, wherein L¹ and L² are —CO—O—, and R¹ and R² are tridecyl,pentadecyl, heptadecyl, nonadecyl, heneicosyl, tricosyl,(Z)-tridec-8-enyl, (Z)-pentadec-8-enyl, (Z)-heptadec-5-enyl,(Z)-heptadec-8-enyl, (E)-heptadec-8-enyl, (Z)-heptadec-10-enyl,(8Z,11Z)-heptadec-8,11-dienyl, (8Z,11Z,14Z)-octadec-8,11,14-trienyl,(Z)-nonadec-10-enyl, (10Z,13 Z)-nonadec-10,13-dienyl,(11Z,14Z)-icos-11,14-dienyl, 2,6,10-trimethylundec-1,5,9-trienyl or2,6,10,14-tetramethylpentadec-1-enyl.
 31. The cationic lipid accordingto any one of claim 28 to 30, wherein a and b are both 0 or
 1. 32. Thecationic lipid according to any one of claim 28 or 29, wherein L³ is asingle bond, R³ is a hydrogen atom, methyl, pyrrolidin-3-yl,piperidin-3-yl, piperidin-4-yl, or alkyl having 1 to 6 carbon atoms oralkenyl having 3 to 6 carbon atoms substituted with 1 to 3substituent(s), which is(are), the same or different, amino,monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidylor morpholinyl, and L¹ and L² are —O—.
 33. The cationic lipid accordingto any one of claim 28 to 30, wherein L³ is —CO— or —CO—O—, R³ ispyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having 1 to 6carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to3 substituent(s), which is(are), the same or different, amino,monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy,carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidylor morpholinyl, wherein at least one of the substituents is amino,monoalkylamino, dialkylamino, trialkylammonio, pyrrolidinyl, piperidylor morpholinyl, and L¹ and L² are identically —CO—O— or —O—CO—.
 34. Thecationic lipid according to any one of claims 28 to 30, wherein R³ is ahydrogen atom, methyl, or alkyl having 1 to 6 carbon atoms or alkenylhaving 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), whichis(are), the same or different, amino, hydroxy or carbamoyl.
 35. Thecationic lipid according to claim 33, wherein R³ is alkyl having 1 to 6carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to3 substituent(s), which is(are), the same or different, amino, hydroxyor carbamoyl.
 36. The cationic lipid according to any one of claims 28to 30, wherein X³ is absent or is methyl.
 37. A composition comprisingthe cationic lipid according to any one of claims 28 to 30, and anucleic acid.
 38. A composition comprising a complex particle of thecationic lipid according to any one of claims 28 to 30 and a nucleicacid, or a complex particle of a nucleic acid and a combination of thecationic lipid and a neutral lipid and/or a polymer.
 39. A compositioncomprising a lipid particle constituted of a complex particle of thecationic lipid according to any one of claims 28 to 30 and a nucleicacid, or a complex particle of a nucleic acid and a combination of thecationic lipid and a neutral lipid and/or a polymer, and a lipidmembrane that encapsulates the complex particle.
 40. The compositionaccording to claim 38, wherein the nucleic acid is a nucleic acid havingan activity of suppressing the expression of the target gene byutilizing RNA interference (RNAi).
 41. The composition according toclaim 40, wherein the target gene is a gene associated with tumor orinflammation.
 42. A method for introducing the nucleic acid into a cellby using the composition according to claim
 38. 43. The method accordingto claim 42, wherein the cell is a cell at a tumor or inflammation siteof a mammal.
 44. The method according to claim 42, wherein the cell is acell in the liver, lungs, kidneys or spleen of a mammal.
 45. The methodaccording to claim 43, wherein the method of the introduction into acell is a method of introduction into a cell by intravenousadministration.
 46. A method for treating cancer or inflammatorydisease, the method including administering the composition according toclaim 41 to a mammal.
 47. The method according to claim 46, wherein themethod of administration is intravenous administration.